Anti-dengue virus antibodies, compositions, methods and uses

ABSTRACT

The present invention relates to at least one novel anti-Dengue virus antibody, including isolated nucleic acids that encode at least one anti-Dengue virus antibody, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e) fromProvisional Application Serial No. 60/443,924 filed on 31 Jan. 2003, theentirety of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to antibodies, including specifiedportions or variants, specific for at least one Dengue virus NS proteinor fragment thereof, as well as nucleic acids encoding such anti-Denguevirus antibodies, complementary nucleic acids, vectors, host cells, andmethods of making and using thereof, including therapeutic formulations,administration and devices.

[0003] Dengue Shock Syndrome (DSS) and dengue hemorrhagic fever (DHF)are caused by one of four closely related, but antigenically distinct,virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4), of the genusFlavivirus. Infection with one of these serotypes does not providecross-protective immunity, so persons living in a dengue-endemic areacan have at least four dengue infections during their lifetimes. Dengueis primarily a disease of the tropics, and the viruses that cause it aremaintained in a cycle that involves humans and Aedes aegypti, adomestic, day-biting mosquito that prefers to feed on humans. Infectionwith dengue viruses produces a spectrum of clinical illness ranging froma nonspecific viral syndrome to severe and fatal hemorrhagic disease.Important risk factors for DHF include the strain and serotype of theinfecting virus, as well as the age, immune status, and geneticpredisposition of the patient.

[0004] Dengue may be the most important mosquito-borne viraldisease-affecting humans; its global distribution is comparable to thatof malaria, and an estimated 2.5 billion people live in areas at riskfor epidemic transmission. Each year, tens of millions of cases ofdengue fever occur and, depending on the year, up to hundreds ofthousands of cases of DHF. The case-fatality rate of DHF in mostcountries is about 5%; most fatal cases are among children and youngadults.

[0005] Accordingly, there is a need to provide new therapeutic,prophylactic and diagnostic agents against Dengue virus. Anti-Denguevirus antibodies or fragments of the present invention provide newtherapeutic and/or prophylactic agents to treat and/or prevent Denguevirus infection and its associated diseases. The anti-Dengue virusantibodies and fragments of the present invention also providediagnostic reagents for detecting infection or epidemiologicalinvestigation in the population.

SUMMARY OF THE INVENTION

[0006] The present invention provides isolated human, primate, rodent,mammalian, chimeric, humanized and/or CDR-grafted anti-Dengue virusantibodies, immunoglobulins, cleavage products and other specifiedportions and variants thereof, as well as anti-Dengue virus antibodycompositions, encoding or complementary nucleic acids, vectors, hostcells, compositions, formulations, devices, transgenic animals,transgenic plants, and methods of making and using thereof, as describedand enabled herein, and in combination with what is known in the art.

[0007] The present invention also provides at least one isolatedanti-Dengue virus antibody as described herein. An antibody according tothe present invention includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulinmolecule, such as but not limited to at least one complementaritydetermining region (CDR) of a heavy or light chain or a ligand bindingportion thereof, a heavy chain or light chain variable region, a heavychain or light chain constant region, a framework region, or any portionthereof, that can be incorporated into an antibody of the presentinvention. An antibody of the invention can include or be derived fromany mammal, such as, but not limited to, a human, a mouse, a rabbit, arat, a rodent, a primate, or any combination thereof, and the like.

[0008] The present invention provides, in one aspect, isolated nucleicacid molecules comprising, complementary, or hybridizing to, apolynucleotide encoding specific anti-Dengue virus antibodies,comprising at least one specified sequence, domain, portion or variantthereof. The present invention further provides recombinant vectorscomprising said anti-Dengue virus antibody nucleic acid molecules, hostcells containing such nucleic acids and/or recombinant vectors, as wellas methods of making and/or using such antibody nucleic acids, vectorsand/or host cells.

[0009] At least one antibody of the invention binds at least onespecified epitope specific to at least one Dengue virus protein,subunit, fragment, portion or any combination thereof, including, butnot limited to, Dengue virus proteins C, preM, E, NS1, NS2A, NS2B, NS3,NS4A, NS4B and NS5. At least one epitope can comprise at least oneantibody binding region that comprises at least one portion of saidprotein, which epitope is preferably comprised of at least 1 to 5 aminoacids of at least one portion thereof, such as but not limited to, atleast one functional, extracellular, soluble, hydrophilic, external orcytoplasmic domain of said protein, or any portion thereof. At least oneantibody of the present invention binds to a Dengue virus NS-1 protein.

[0010] At least one antibody can optionally comprise at least onespecified portion of at least one complementarity determining region(CDR) (e.g., CDR1, CDR2 or CDR3 of the heavy or light chain variableregion) and/or at least one constant or variable framework region or anyportion thereof. The at least one antibody amino acid sequence canfurther optionally comprise at least one specified substitution,insertion or deletion as described herein or as known in the art.

[0011] The present invention also provides at least one isolatedanti-Dengue virus antibody as described herein, wherein the antibody hasat least one activity, such as, but not limited to, inhibition orenhancement of Dengue virus-induced cell death or damage resulting fromactivity of the host's immune system, neutralization of a biologicalactivity of a Dengue virus NS protein, inhibition of Dengue virusreplication, preventing Dengue virus-related disease in a patient,mitigating symptoms of Dengue virus infection in a patient, diagnosingthe presence of Dengue virus in a sample, and epidemiological analysis.A(n) anti-Dengue virus antibody can thus be screened for a correspondingactivity according to known methods, such as but not limited to, atleast one biological activity towards a Dengue virus protein.

[0012] The present invention also provides at least one method forexpressing at least one anti-Dengue virus antibody, in a host cell,comprising culturing a host cell as described herein under conditionswherein anti-Dengue virus antibody is expressed in detectable and/orrecoverable amounts.

[0013] The present invention also provides at least one compositioncomprising: a) an isolated anti-Dengue virus antibody encoding nucleicacid and/or antibody as described herein; and b) a suitable carrier ordiluent. The carrier or diluent can optionally be pharmaceuticallyacceptable, according to known carriers or diluents. The composition canoptionally further comprise at least one further compound, protein orcomposition.

[0014] The present invention further provides at least one anti-Denguevirus antibody method or composition, for administering atherapeutically effective amount to modulate or treat at least oneDengue virus related condition in a cell, tissue, organ, animal orpatient and/or, prior to, subsequent to, or during a related condition,as known in the art and/or as described herein.

[0015] The present invention also provides at least one composition,device and/or method of delivery of a therapeutically orprophylactically effective amount of at least one anti-Dengue virusantibody, according to the present invention.

[0016] The present invention further provides at least one anti-Denguevirus antibody method or composition, for diagnosing at least one Denguevirus related condition in a cell, tissue, organ, animal, patient orpopulation of subjects, and/or, prior to, subsequent to, or during arelated condition, as known in the art and/or as described herein.

[0017] The present invention also provides at least one composition,device and/or method of delivery for diagnosing the presence or absenceof at least one anti-Dengue virus antibody in a sample, according to thepresent invention.

DESCRIPTION OF THE FIGURES

[0018]FIG. 1, Panels A, B and C: Shows a graphical representationshowing an assay for ability of anti-Dengue virus antibody to inhibitDengue virus infectivity. FIG. 1, Panel A shows a titration of Dengueantigen against IgG DEN3 mAb and Fab Sid33. FIG. 1, Panel B shows atitration of Dengue antigen against Sid33 Fab at 20 μl/mL. FIG. 1, PanelC shows a titration of Fab Sid33 and IgG DEN3 antibodies against Dengueantigen at (1:500). Based on data from antigen titration, peak bindingoccurred at approximately (1:500) dilution of Dengue antigen. Thus,antibodies were titrated against the antigen at (1:500) beginning at 25μg/mL to create a binding curve.

[0019]FIG. 2: Shows DNA sequences of the anti-Dengue virus mAb heavychain variable regions (SEQ ID NO: 1).

[0020]FIG. 3: Shows DNA sequences of the anti-Dengue virus mAb lightchain variable regions (SEQ ID NO: 2).

[0021]FIG. 4: Shows deduced amino acid sequences of the anti-Denguevirus mAb heavy chain variable regions. The amino acid sequences shown(single letter abbreviations) were deduced from DNA sequence. The aminosequences are shown partitioned into the framework (FR) andcomplementarity determining region (CDR) domains (SEQ ID NO: 3).

[0022]FIG. 5: Shows deduced amino acid sequences of the anti-Denguevirus mAb light chain variable regions. The amino acid sequences shown(single letter abbreviations) were deduced from DNA sequence. The aminosequences are shown partitioned into the framework (FR) andcomplementarity determining region (CDR) domains (SEQ ID NO: 4).

[0023]FIG. 6: Shows schematic illustrations of the heavy and light chainexpression plasmid pDR1 11070 (bp) used to make the anti-Dengue virusantibody-expressing cells.

DESCRIPTION OF THE INVENTION

[0024] The present invention provides isolated, anti-Dengue virusantibodies, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding at least one anti-Denguevirus antibody. The present invention further includes, but is notlimited to, methods of making and using such nucleic acids andantibodies, including diagnostic and therapeutic compositions, methodsand devices.

[0025] Dengue viruses are 50 nm spherical enveloped particles withicosahedral capsid symmetry. Virions incorporate a single strand ofpositive-sense RNA of approximately 11,000 bases. The RNA is packagedwithin a capsid (C) protein. The surrounding shell is composed of 90copies of dimeric envelope (E) proteins and interspersed matrix (M)proteins. Fifteen to twenty percent of virion weight is made up oflipids incorporated from host cell membranes; 9 to 10% of virion weightis in glycolipids or glycoproteins.¹

[0026] Dengue viruses belong to the Flavivirus genus, some 68antigenically related viruses whose genes, organized from the 5′ end,code for 10 proteins, C, pre-M, E, NS-1 (non-structural), NS2A, NS2B,NS3, NS4A, NS4B, and NS5. Most flaviviruses are transmitted betweenvertebrate hosts by blood-sucking arthropods. 20 [0027] There are fourDengue viruses, Dengue (DEN)-1, -2, -3, and -4. These evolved insub-human primates from a common ancestor and have been transmitted inan urban cycle, Aedes aegypti—humans—Aedes aegypti, for around onethousand years. 2

[0027] Dengue viruses share 60+% genome within the Dengue subgroup.Thus, shared antigens are expressed on virion surfaces or the surface ofinfected cells. Primary infections raise common Dengue antibodies,readily detected by the ELISA and hemagglutination-inhibition tests, butless well detected by plaque reduction neutralization tests (PRNT). PRNTare performed in a wide range of continuous cell lines.^(3, 4, 5)

[0028] As described by Sabin (1952), adult human volunteers infectedwith DEN-1 or DEN-2 viruses were protected from clinical illness whenchallenged with heterologous virus within two months of primaryinfections. However, viremic infections occurred in mono-immunevolunteers challenged with heterologous virus between two and threemonths after primary infection. These infections “gave rise to malaiseand slight fever.” Mild illnesses accompanied cross-challenges up tonine months following primary infection and unaltered illnessesthereafter.⁶ Other than the studies of Sabin, cross protection amongvarious Dengue viruses and strains has not been studied systematically.Third and fourth Dengue infections are documented in prospectiveseroepidemiological studies, but seldom result in clinical illness.⁷

[0029] The strongest evidence that Dengue antibodies both protectagainst and enhance Dengue virus infections is the regular occurrence ofsevere DHF in infants during their first Dengue infection.^(8, 9, 10)This group comprises 5% to 10% of all DHF cases and at peak risk period(eight months) infants are four times more likely to develop DHF duringa first Dengue infection than are older children during second dengueinfections. Initially, infants are completely protected against Dengueillnesses.¹¹ As maternal antibodies wane below protective threshold(thought to be around 1:10 in a conventional PRNT), infants become atrisk to enhanced infections and disease.^(9, 12) These infants aresusceptible to infection by any Dengue virus type.

[0030] This phenomenon has been reproduced experimentally in an in vivomodel. Enhanced DEN-2 viremias compared with control animals wereobserved regularly in five rhesus monkeys infected immediately followingintravenous inoculation of small quantities of human cord blood-deriveddengue antibodies.¹³

[0031] Evidence that DHF/DSS occurs at high frequency during secondaryDengue infections comes from a multitude of sources over more than 40years of observation. Four prospective studies have documentedhospitalized Dengue only among children with pre-hospitalization Dengueantibodies.^(14, 15, 16, 17)

[0032] Enhanced viremias during secondary DEN-2 infections have beenobserved in vivo in rhesus monkeys infected in the sequences DEN-1followed by DEN-2, DEN-3 followed by DEN-2, and DEN-4 followed byDEN-2.¹⁸ Enhanced viremias have not been observed in monkeys infected inother sequences (all nine other sequences have been tried). However,only limited numbers of monkeys were infected in other sequences.

[0033] Enhanced viremias, or Antibody-dependent enhancements (ADE),during secondary infections predict (and correlate with) diseaseseverity during secondary dengue infections in children.¹⁹

[0034] ADE can be demonstrated and measured in vitro using as host cellsa wide range of primary blood leukocyte and FcR-bearing cell cultures.²⁰

[0035] Critical site-specific monoclonal antibodies neutralize at lowdilutions and enhance at high dilutions. Some group-reactive monoclonalantibodies neutralize at low dilutions and enhance at highdilutions.^(20, 21, 22, 23)

[0036] Cross-reactive neutralizing antibodies are often observedfollowing a single Dengue virus infection and more often inDengue-infected Japanese encephalitis immunes.^(4,17,24) Despiteheterologous antibodies (measured in vitro) second Dengue virusinfections occur regularly, only rarely symptomatic. In a 1980 cohortstudy, inapparent infections were observed in 85% of forty Bangkokschool children who experienced secondary DEN-2 infections. Undilutedpre-infection serum from these children prevented DEN-2 infection incultures of human monocytes. But, undiluted sera from six of the sevencohort children who experienced severe illness did not neutralize DEN-2;ADE was observed.²⁴ More recently, children acquired clinically overtsecondary DEN-3 infections when their pre-illness sera containedheterologous DEN-3 neutralizing antibodies. Use of the autologous DEN-3strain (virus isolated from patient) significantly reduced neutralizingantibody titers compared with use of laboratory-passaged “standard”strains. The severity of secondary DEN-3 infections was inverselyrelated to the neutralizing antibody titers against autologous DEN-3strains.

[0037] The most dramatic illustration of infection down-regulation byheterotypic antibodies is the non-“virulence” of the American genotypeDEN-2. When this virus circulated in Peru in a population partiallyimmune to DEN-1, no DHF was observed.²⁵ But, the capacity of humananti-DEN-1 sera to neutralize different DEN-2 viruses was not the same:all American genotype DEN-2 viruses were highly neutralized byanti-DEN-1, while anti-DEN-1 poorly neutralized (DHF-producing) SE AsianDEN-2 strains.²⁶ Asian DEN-2 strains, chronically circulating with otherDEN viruses may have been selected to “escape” neutralization byheterotypic antibodies.

[0038] A more rapid “escape” phenomenon has been observed. Rapid(month-to-month) disease severity increases were observed during twoepidemics in which monotypic DEN-2 was transmitted in humans immune toDEN-1.²⁷

[0039] Affinity maturation of antibody responses occurs followingflavivirus infections. This was demonstrated by rising logneutralization indices over a four-year follow-up of American servicemenwho were subclinically infected with Japanese Encephalitis Virus. Theywere bled at one and five years after infection.²⁸ Affinity maturationoffers an explanation of the observation that secondary DEN-2 infectionsat an interval of twenty years after primary DEN-1 were much more severethan when the same genotype viruses resulted in secondary infections atan interval of four years. This could be the result of antibody becomingmore avidly directed at type-specific neutralization site(s) withcorrespondingly less broad reactivity, permitting ADE to occur.²⁹

[0040] As used herein, an “Anti-Dengue virus antibody,” “Anti-Denguevirus antibody portion,” or “Anti-Dengue virus antibody fragment” and/or“Anti-Dengue virus antibody variant” and the like include any protein orpeptide containing molecule that comprises at least a portion of animmunoglobulin molecule, such as, but not limited to, at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, or at least one portion of a Denguevirus protein, which can be incorporated into an antibody of the presentinvention. Such antibody optionally further affects a specific ligand,such as, but not limited to, where such antibody modulates, decreases,increases, antagonizes, angonizes, mitigates, alleviates, blocks,inhibits, abrogates and/or interferes with at least one Dengue virusinfectivity activity or replication, or with Dengue virus cellularreceptor activity or binding, in vitro, in situ and/or in vivo. As anon-limiting example, a suitable anti-Dengue virus antibody, specifiedportion or variant of the present invention can bind at least one Denguevirus, or specified portions, variants or domains thereof. A suitableanti-Dengue virus antibody, specified portion, or variant can alsooptionally affect at least one of Dengue virus activity or function,such as, but not limited to, RNA, DNA or protein synthesis, Dengue virusinfectivity, Dengue virus release, Dengue virus receptor signaling,Dengue virus replication, Dengue virus production and/or synthesis andfacilitating the destruction or recognition of Dengue virus infectedcells by the host immune system. The term “antibody” is further intendedto encompass antibodies, digestion fragments, specified portions andvariants thereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian Dengue virus. For example, antibodyfragments capable of binding to Dengue virus or portions thereof,including, but not limited to Fab (e.g., by papain digestion), Fab′(e.g., by pepsin digestion and partial reduction) and F(ab′)₂ (e.g., bypepsin digestion), facb (e.g., by plasmin digestion), pFc′ (e.g., bypepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partialreduction and re-aggregation), Fv or scFv (e.g., by molecular biologytechniques) fragments, are encompassed by the invention (see, e.g.,Colligan, Immunology, supra).

[0041] Such fragments can be produced by enzymatic cleavage, syntheticor recombinant techniques, as known in the art and/or as describedherein. Antibodies can also be produced in a variety of truncated formsusing antibody genes in which one or more stop codons have beenintroduced upstream of the natural stop site. For example, a combinationgene encoding a F(ab′)₂ heavy chain portion can be designed to includeDNA sequences encoding the CH, domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

[0042] As used herein, the term “human antibody” refers to an antibodyin which substantially every part of the protein (e.g., CDR, framework,C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L),V_(H))) is substantially non-immunogenic in humans, with only minorsequence changes or variations. Similarly, antibodies designated primate(monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guineapig, hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain) genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

[0043] Bispecific, heterospecific, heteroconjugate or similar antibodiescan also be used that are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one Dengue virus protein, the other one is for any otherantigen. Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, 305 NATURE, 537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668; 6,193,967; 6,132,992; 6,106,833; 6,060,285; 6,037,453;6,010,902; 5,989,530; 5,959,084; 5,959,083; 5,932,448; 5,833,985;5,821,333; 5,807,706; 5,643,759; 5,601,819; 5,582,996; 5,496,549;4,676,980; WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., 10EMBO J., 3655 (1991), Suresh et al., 121 METHODS IN ENZYMOLOGY, 210(1986), each entirely incorporated herein by reference.

[0044] Anti-Dengue virus antibodies (also termed Dengue virusantibodies) useful in the methods and compositions of the presentinvention can optionally be characterized by high affinity binding toDengue virus and optionally and preferably having low toxicity. Inparticular, an antibody, specified fragment or variant of the invention,where the individual components, such as the variable region, constantregion and framework, individually and/or collectively, optionally andpreferably possess low immunogenicity, is useful in the presentinvention. The antibodies that can be used in the invention areoptionally characterized by their ability to treat patients for extendedperiods with measurable alleviation of symptoms and low and/oracceptable toxicity. Low or acceptable immunogenicity and/or highaffinity, as well as other suitable properties, can contribute to thetherapeutic results achieved. (Elliott et al., 344 LANCET, 1125-1127(1994), entirely incorporated herein by reference).

[0045] Utility

[0046] The isolated nucleic acids of the present invention can be usedfor production of at least one anti-Dengue virus antibody or specifiedvariant thereof, which can be used to measure or effect in a cell,tissue, organ or animal (including mammals and humans), to diagnose,monitor, modulate, treat, alleviate, help prevent the incidence of, orreduce the symptoms of, at least one Dengue virus condition, selectedfrom, but not limited to, at least one of an immune disorder or disease,a cardiovascular disorder or disease, an infectious, malignant, and/orneurologic disorder or disease.

[0047] Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-Dengue virus antibody to a cell, tissue, organ, animal or patientin need of such modulation, treatment, alleviation, prevention, orreduction in symptoms, effects or mechanisms. The effective amount cancomprise an amount of about 0.001 to 50 mg/kg per single (e.g., bolus),multiple or continuous administration, or to achieve a serumconcentration of 0.01 to 500 μg/ml serum concentration per single,multiple, or continuous administration, or any effective range or valuetherein, as done and determined using known methods, as described hereinor known in the relevant arts.

[0048] Citations

[0049] All publications or patents cited herein are entirelyincorporated herein by reference as they show the state of the art atthe time of the present invention and/or to provide description andenablement of the present invention. Publications refer to anyscientific or patent publications, or any other information available inany media format, including all recorded, electronic or printed formats.The following references are entirely incorporated herein by reference:Ausubel et al. (Ed.), Current Protocols in Molecular Biology, (JohnWiley & Sons, Inc., New York, N.Y. (1987-1991)); Sambrook et al.,Molecular Cloning: A Laboratory Manual 2^(nd) Edition, (Cold SpringHarbor, N.Y. (1989)); Harlow and Lane, Antibodies, A Laboratory Manual,(Cold Spring Harbor, N.Y. (1989)); Colligan et al. (Eds.), CurrentProtocols in Immunology, (John Wiley & Sons, Inc., NY (1994-2001));Colligan et al., Current Protocols in Protein Science, (John Wiley &Sons, NY, N.Y., (1997-2001)).

[0050] Antibodies of the Present Invention

[0051] At least one anti-Dengue virus antibody of the present inventioncan be optionally produced by a cell line, a mixed cell line, animmortalized cell or clonal population of immortalized cells, as wellknown in the art. See, e.g., Ausubel et al. (Ed.), Current Protocols inMolecular Biology, (John Wiley & Sons, Inc., New York, N.Y.(1987-2001)); Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, (Cold Spring Harbor, N.Y. (1989)); Harlow and Lane,Antibodies, A Laboratory Manual, (Cold Spring Harbor, N.Y. (1989));Colligan, et al. (Eds.), Current Protocols in Immunology, (John Wiley &Sons, Inc., NY (1994-2001)); Colligan et al., Current Protocols inProtein Science, (John Wiley & Sons, NY, N.Y., (1997-2001)), eachentirely incorporated herein by reference.

[0052] Human antibodies that are specific for Dengue virus proteins orfragments thereof can be raised against an appropriate immunogenicantigen, such as isolated Dengue virus protein or a portion thereof(including synthetic molecules, such as synthetic peptides). Otherspecific or general mammalian antibodies can be similarly raised.Preparation of immunogenic antigens, and monoclonal antibody productioncan be performed using any suitable technique.

[0053] In one approach, a hybridoma is produced by fusing a suitableimmortal cell line (e.g., a myeloma cell line) such as, but not limitedto, Sp2/0, Sp2/0-AG14, P3/NS1/Ag4-1, P3X63Ag8.653, MCP-11, S-194, or thelike, or heteromyelomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com, and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, Chapter 2,entirely incorporated herein by reference.

[0054] Antibody producing cells can also be obtained from the peripheralblood or, preferably the spleen or lymph nodes, of humans or othersuitable animals that have been immunized with the antigen of interest.Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an antibody, specifiedfragment or variant thereof, of the present invention. The fused cells(hybridomas) or recombinant cells can be isolated using selectiveculture conditions or other suitable known methods, and cloned bylimiting dilution or cell sorting, or other known methods. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay (e.g., ELISA).

[0055] Other suitable methods of producing or isolating antibodies ofthe requisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684; PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO 90/14443; WO 90/14424; WO 90/14430;PCT/US94/1234; WO 92/18619; WO 96/07754 (Scripps); EP 614989(MorphoSys); WO 95/16027 (BioInvent); WO 88/06630; WO 90/3809 (Dyax);U.S. Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO 89/06283;EP 371998; EP 550400; (Xoma); EP 229046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862; WO 86/05803; EP590689 (Ixsys, now known as Applied Molecular Evolution (AME), eachentirely incorporated herein by reference) or that rely uponimmunization of transgenic animals (e.g., SCID mice, Nguyen et al., 41MICROBIOL. IMMUNOL., 901-907 (1997); Sandhu et al., 16 CRIT. REV.BIOTECHNOL., 95-118 (1996); Eren et al., 93 IMMUNOL., 154-161 (1998),each entirely incorporated by reference as well as related patents andapplications) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal., 94 PROC. NATL. ACAD. SCI. USA, 4937-4942 (May, 1997); Hanes et al.,95 PROC. NATL. ACAD. SCI. USA, 14130-14135 (November, 1998); single cellantibody producing technologies (e.g., selected lymphocyte antibodymethod (“SLAM”) (U.S. Pat. No. 5,627,052; Wen et al., 17 J. IMMUNOL.,887-892 (1987); Babcook et al., 93 PROC. NATL. ACAD. SCI. USA, 7843-7848(1996); gel microdroplet and flow cytometry (Powell et al., 8BIOTECHNOL., 333-337 (1990); One Cell Systems, Cambridge, Mass.; Gray etal., 182 J. IMM. METH., 155-163 (1995); Kenny et al., 13 BIO/TECHNOL.,787-790 (1995); B-cell selection (Steenbakkers et al., 19 MOLEC. BIOL.REPORTS, 125-134 (1994); Jonak et al., Progress Biotech Vol. 5 In VitroImmunization in Hybridoma Technology, (Borrebaeck (Ed.), ElsevierScience Publishers B. V., Amsterdam, Netherlands (1988)).

[0056] Methods for engineering or humanizing non-human or humanantibodies can also be used and are well known in the art. Generally, ahumanized or engineered antibody has one or more amino acid residuesfrom a source which is non-human, e.g., but not limited to, mouse, rat,rabbit, non-human primate or other mammal. These human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable, constant or other domain of a knownhuman sequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/guerv.fcgi www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/research_tools.html,www.mgen.uniheidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/mrc7/mikeimages.html; www.antibody resource.com/;mcb.harvard.edu/BioLinks/Immunology.html.; www.immunologylink.com/;pathbox.wustl.edu/˜hcenter/index.html; www.biotech.ufl.edu/˜hcl/;www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehimeu.ac.jp/˜yasuhito/Elisa.html, www.biodesign.com/table.asp;www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/˜fccl/protocol.html; www.isacnet.org/sites_geo.html;aximt1.imt.uni-marburg.de/˜rek/AEPStart.html;baserv.uci.kun.nl/˜jraats/liniks1.htmlwww.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrccpe.cam.ac.uk/imtdoc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/˜martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/www abgen.html;www.unizh.ch/˜honegger/AHOseminar/Slide01.html,www.cryst.bbk.ac.uk/˜ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgi/index.html;wwwcryt.bioc.cam.ac.uk/˜fmolina/Webpages/Pept/spottech.html;www.jerini.de/fr₁₃ products.htm; www.patents.ibm.com/ibm.html; Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

[0057] Such imported sequences can be used to reduce immunogenicity orreduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Humanization or engineering of antibodiesof the present invention can be performed using any known method, suchas but not limited to those described in, Winter (Jones et al., 321NATURE, 522 (1986); Riechmann et al., 332 NATURE, 323 (1988); Verhoeyenet al., 239 SCIENCE, 1534 (1988); Sims et al., 151 J. IMMUNOL., 2296(1993); Chothia and Lesk, 196 J. MOL. BIOL., 901 (1987); Carter et al.,89 PROC. NATL. ACAD. SCI. U.S.A., 4285 (1992); Presta et al., 151 J.IMMUNOL., 2623 (1993); U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514;5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352;6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539;4,816,567; International Application Nos.: PCT/US98/16280;PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234;PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; InternationalPublication Nos.: WO 90/14443; WO 90/14424; WO 90/14430; and EuropeanPatent Application No. EP 229246, each entirely incorporated herein byreference, including references cited therein.

[0058] The anti-Dengue virus antibody can also be optionally generatedby immunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-Dengue virus antibody can be isolated fromsuch animals and immortalized using suitable methods, such as themethods described herein.

[0059] Transgenic mice that can produce a repertoire of human antibodiesthat bind to human antigens and other foreign antigens can be producedby known methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428;5,569,825; 5,545,806; 5,625,126; 5,625,825; 5,633,425; 5,661,016 and5,789,650 issued to Lonberg et al.; Jakobovits et al., WO 98/50433;Jakobovits et al., WO 98/24893; Lonberg et al., WO 98/24884; Lonberg etal., WO 97/13852; Lonberg et al., WO 94/25585; Kucherlapate et al., WO96/34096; Kucherlapate et al., EP 0463151 B1; Kucherlapate et al., EP0710719 A1; Surani et al., U.S. Pat. No. 5,545,807; Bruggemann et al.,WO 90/04036; Bruggemann et al., EP 0438474 B1; Lonberg et al., EP0814259 A2; Lonberg et al., GB 2272440 A; Lonberg et al., 368 NATURE,856-859 (1994); Taylor et al., 6(4) INT. IMMUNOL., 579-591 (1994); Greenet al, 7 NATURE GENETICS, 13-21 (1994); Mendez et al., 15 NATUREGENETICS, 146-156 (1997); Taylor et al., 20(23) NUCLEIC ACIDS RESEARCH,6287-6295 (1992); Tuaillon et al., 90(8) PROC. NATL. ACAD. SCI. USA,3720-3724 (1993); Lonberg et al., 13(1) INT. REV. IMMUNOL., 65-93 (1995)and Fishwald et al., 14(7) NAT BIOTECHNOL, 845-851 (1996), which areeach entirely incorporated herein by reference). Generally, these micecomprise at least one transgene comprising DNA from at least one humanimmunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

[0060] The antigenic specificity of antibodies, for example those madeby the methods described herein, can be conveniently determined usingtechniques known in the art, including, but not limited to, randompeptide display libraries. This method involves the screening of largecollections of peptides for individual members that are recognized bythe antibody. Antibody screening of peptide display libraries is wellknown in the art. The displayed random peptide sequences can be from 3to 5000 or more amino acids in length, frequently from 5 to 100 aminoacids long, and often from about 8 to 25 amino acids long. In additionto direct chemical synthetic methods for generating peptide libraries,several recombinant DNA methods have been described. One type involvesthe display of random peptide sequences on the surface of abacteriophage or cell. Each bacteriophage or cell contains thenucleotide sequence encoding the particular displayed peptide sequence.Antibody is immobilized on a substrate and incubated with bacteriophageor cells bearing the random peptide library on their surface. Afterseveral rounds of selection by panning as described for example in Lu etal., 13 BIO/TECHNOLOGY, 366-372 (1995), which is incorporated byreference herein, bacteriophage colonies are sequenced to determine thecommon peptide sequence recognized by the antibody. This method allowsthe identification of the antigen recognition sequence for the antibody.Such methods are described in PCT Patent Publication Nos. WO 91/18980,WO 91/19818, and WO 93/08278.

[0061] Another method well known in the art for identifying antibodiesthat are specific for a particular antigen is to utilize virus,bacteriophage or host cells expressing antibody molecules on theirsurface. In this method, DNA encoding the antibody or antibody fragmentis contained within the virus, bacteriophage, host cell or otherreplication competent system. Portions of antibody molecules areexpressed on the surface of the virus, bacteriophage, host cell or otherreplication competent system and are selected by binding to immobilizedantigen. After several rounds of selection, the DNA encoding theantibody or antibody fragment is isolated. See PCT Patent PublicationNo. WO 91/17271. Other systems for generating libraries of random andspecific peptides have aspects of both in vitro chemical synthesis andrecombinant methods. See, PCT Patent Publication Nos. WO 92/05258, WO92/14843, and WO 96/19256. See also, U.S. Pat. Nos. 5,658,754 and5,643,768. Random peptide display libraries, antibody fragment displaylibraries, vectors, and screening kits for performing these methods arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge Antibody Technologies (Cambridgeshire, UK). See,e.g., U.S. Pat. Nos. 4,704,692; 4,874,702; 4,939,666; 4,946,778;5,260,203; 5,455,030; 5,518,889; 5,534,621; 5,656,730; 5,763,733;5,767,260; 5,856,456 are assigned to Enzon, 5,223,409; 5,403,484;5,571,698 and 5,837,500 are assigned to Dyax; 5,427,908 and 5,580,717are assigned to Affymax; 5,885,793 is assigned to Cambridge AntibodyTechnologies; 5,750,373 is assigned to Genentech; 5,618,920; 5,595,898;5,576,195; 5,698,435; 5,693,493 and 5,698,417 are assigned to Xoma;Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the abovepatents and publications entirely incorporated herein by reference.

[0062] Antibodies of the present invention can also be prepared using atleast one anti-Dengue virus antibody-encoding nucleic acid to providetransgenic animals or mammals, such as goats, cows, horses, sheep, andthe like, that produce such antibodies in their milk. Such animals canbe provided using known methods. See, e.g., but not limited to, U.S.Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616;5,565,362 and 5,304,489, and the like, each of which is entirelyincorporated herein by reference.

[0063] Antibodies of the present invention can additionally be preparedusing at least one anti-Dengue virus antibody-encoding nucleic acid toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco and maize) that produce such antibodies, specifiedportions or variants in the plant parts or in cells cultured therefrom.As a non-limiting example, transgenic tobacco leaves expressingrecombinant proteins have been successfully used to provide largeamounts of recombinant proteins, e.g., using an inducible promoter. See,e.g., Cramer et al., 240 CURR. TOP. MICROBOL. IMMUNOL., 95-118 (1999)and references cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., 464ADV. EXP. MED. BIOL., 127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., 38 PLANT MOL. Biol., 101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to known methods. See also,e.g., Fischer et al., 30 BIOTECHNOL. APPL. BIOCHEM., 99-108 (October,1999), Ma et al., 13 TRENDS BIOTECHNOL., 522-527 (1995); Ma et al., 109PLANT PHYSIOL., 341-346 (1995); Whitelam et al., 22 BIOCHEM. SOC.TRANS., 940-944 (1994); and references cited therein. Each of the abovereferences is entirely incorporated herein by reference.

[0064] The antibodies of the invention can bind Dengue virus proteinswith a wide range of affinities (K_(D)). In a preferred embodiment, atleast one human mAb of the present invention can optionally bind Denguevirus NS1 protein with high affinity. For example, a human mAb can bindDengue virus NS1 protein with a K_(D) equal to or less than about 10⁻⁷M, such as, but not limited to, 0.1 to 9.9 (or any range or valuetherein) X 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹² or any range or valuetherein.

[0065] The affinity or avidity of an antibody for an antigen can bedetermined experimentally using any suitable method. (See, for example,Berzofsky et al., “Antibody-Antigen Interactions,” In FundamentalImmunology, (W. E. Paul (Ed.), Raven Press: New York, N.Y., 1984); JanisKuby, Immunology, (W. H. Freeman and Company: New York, N.Y., 1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

[0066] [0064] The ability of the anti-Dengue virus antibodies describedherein may also be tested for their ability to neutralize Dengue virususing virus neutralization procedures well known to those of ordinaryskill in the art. Such virus neutralization procedures include, but arenot limited to, those described in R. Rico-Hesse, “Molecular Evolutionand Distribution of Dengue Viruses Type 1 and 2 in Nature”, 174VIROLOGY, 479-493 (1990); B. L. Innis, “Antibody Responses to DengueVirus Infection”, Dengue and Dengue Hemorrhagic Fever, (D. J. Gubler andG. Kuno (Eds.), CAB International, Cambridge (1997)) 221-243; Innis etal., “An Enzyme-Linked Immunosorbent Assay to Characterize DengueInfections Where Dengue and Japanese Encephalitis Co-Circulate”, 40 AM.J. TROP. MED. HYG., 418-427 (1989); and Kochel et al., “Neutralizationof American Genotype Dengue 2 Viral Infection by Dengue 1 Antibodies MayHave Prevented Dengue Hemorrhagic Fever in Iquitos, Peru”, LANCET(2002), which are each incorporated by reference herein.

[0067] Nucleic Acid Molecules

[0068] Using the information provided herein, such as the nucleotidesequences encoding at least 70% to 100% of the contiguous amino acids ofat least one of SEQ ID NOS: 3 and 4, specified fragments, variants orconsensus sequences thereof, or a suitable vector comprising at leastone of these sequences, a nucleic acid molecule of the present inventionencoding at least one anti-Dengue virus antibody can be obtained usingmethods described herein or as known in the art.

[0069] Nucleic acid molecules of the present invention can be in theform of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the formof DNA, including, but not limited to, cDNA and genomic DNA obtained bycloning or produced synthetically, or any combinations thereof. The DNAcan be double-stranded or single-stranded, or any combination thereof.Any portion of at least one strand of the DNA or RNA can be the codingstrand, also known as the sense strand, or it can be the non-codingstrand, also referred to as the anti-sense strand.

[0070] Isolated nucleic acid molecules of the present invention caninclude nucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NO: 1) or light chain (e.g., SEQID NO: 2); nucleic acid molecules comprising the coding sequence for ananti-Dengue virus antibody or variable region (e.g., SEQ ID NOS: 3 and4); and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-Denguevirus antibody as described herein and/or as known in the art. Ofcourse, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate nucleicacid variants that code for specific anti-Dengue virus antibodies of thepresent invention. See, e.g., Ausubel et al., supra, and such nucleicacid variants are included in the present invention. Non-limitingexamples of isolated nucleic acid molecules of the present inventioninclude SEQ ID NOS: 1 and 2.

[0071] As indicated herein, nucleic acid molecules of the presentinvention which comprise a nucleic acid encoding an anti-Dengue virusantibody can include, but are not limited to, those encoding the aminoacid sequence of an antibody fragment, by itself; the coding sequencefor the entire antibody or a portion thereof; the coding sequence for anantibody, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including, but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example—ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding an antibody can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedantibody comprising an antibody fragment or portion.

[0072] Construction of Nucleic Acids

[0073] The isolated nucleic acids of the present invention can be madeusing (a) recombinant methods, (b) synthetic techniques, (c)purification techniques, or combinations thereof, as well-known in theart.

[0074] The nucleic acids can conveniently comprise sequences in additionto a polynucleotide of the present invention. For example, amulti-cloning site comprising one or more endonuclease restriction sitescan be inserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

[0075] Additional sequences can be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Use of cloning vectors,expression vectors, adapters, and linkers is well known in the art.(See, e.g., Ausubel, supra; or Sambrook, supra)

[0076] Recombinant Methods for Constructing Nucleic Acids

[0077] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

[0078] Synthetic Methods for Constructing Nucleic Acids

[0079] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by known methods (see, e.g.,Ausubel et al., supra). Chemical synthesis generally produces asingle-stranded oligonucleotide, which can be converted intodouble-stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. One of skill in the art will recognize that while chemicalsynthesis of DNA can be limited to sequences of about 100 or more bases,longer sequences can be obtained by the ligation of shorter sequences.

[0080] Recombinant Expression Cassettes

[0081] The present invention further provides recombinant expressioncassettes comprising a nucleic acid of the present invention. A nucleicacid sequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

[0082] In some embodiments, isolated nucleic acids that serve aspromoter, enhancer, or other elements can be introduced in theappropriate position (upstream, downstream or in intron) of anon-heterologous form of a polynucleotide of the present invention so asto up or down regulate expression of a polynucleotide of the presentinvention. For example, endogenous promoters can be altered in vivo orin vitro by mutation, deletion and/or substitution.

[0083] Vectors And Host Cells

[0084] The present invention also relates to vectors that includeisolated nucleic acid molecules of the present invention, host cellsthat are genetically engineered with the recombinant vectors, and theproduction of at least one anti-Dengue virus antibody by recombinanttechniques, as is well known in the art. See, e.g., Sambrook et al.,supra; Ausubel et al., supra, each entirely incorporated herein byreference.

[0085] The polynucleotides can optionally be joined to a vectorcontaining a selectable marker for propagation in a host. Generally, aplasmid vector is introduced in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid. If thevector is a virus, it can be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

[0086] The DNA insert should be operatively linked to an appropriatepromoter. The expression constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, aribosome-binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

[0087] Expression vectors will preferably but optionally include atleast one selectable marker. Such markers include, e.g., but not limitedto, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636 and 5,179,017;ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359 and 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0088] At least one antibody of the present invention can be expressedin a modified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

[0089] Those of ordinary skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention.

[0090] Alternatively, nucleic acids of the present invention can beexpressed in a host cell by turning on (by manipulation) expression in ahost cell that contains endogenous DNA encoding an antibody of thepresent invention. Such methods are well known in the art, e.g., asdescribed in U.S. Pat. Nos. 5,580,734; 5,641,670; 5,733,746 and5,733,761, entirely incorporated herein by reference.

[0091] Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). In one embodiment, host cells include cells oflymphoid origin such as myeloma and lymphoma cells.

[0092] Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062 and 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

[0093] When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague et al., 45 J.VIROL., 773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

[0094] Purification of an Antibody

[0095] An anti-Dengue virus antibody can be recovered and purified fromrecombinant cell cultures by well-known methods including, but notlimited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, (John Wiley & Sons,New York, N.Y., 1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, eachentirely incorporated herein by reference.

[0096] Antibodies of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacteria, fungiard, yeast, plant, insect,non-mammalian, and mammalian cells. Depending upon the host employed ina recombinant production procedure, the antibody of the presentinvention can be glycosylated or can be non-glycosylated, withglycosylated in certain embodiments. Such methods are described in manystandard laboratory manuals, such as Sambrook, supra, Sections17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20,Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

[0097] Anti-Dengue Virus Antibodies

[0098] The isolated antibodies of the present invention comprise anantibody amino acid sequences disclosed herein encoded by any suitablepolynucleotide, or any isolated or prepared antibody. The human antibodyor antigen-binding fragment binds Dengue virus NS protein and therebypartially or substantially neutralizes at least one biological activityof the NS protein, or it may interfere with the replication cycle of thevirus, or it may enhance the recognition of virus-infected cells by thehost immune system which may facilitate killing of virus-infected cellsand clearance of viral infection. The antibody of the present inventionis not expected to cause the observed enhancement of Dengue virusinfection as seen with antibodies that are specific for Denguevirus.^(20, 21, 22, 23) An antibody, or specified portion or variantthereof, may partially or substantially neutralize at least onebiological activity of at least one Dengue virus NS protein or fragment.The antibody may also bind the viral NS protein or fragment and therebyinhibit activities mediated through Dengue virus NS protein-dependent orNS protein-mediated mechanisms. Dengue virus infected cells present ontheir surface the Dengue virus NS proteins. In addition, since Denguevirus is an enveloped virus, these proteins may also be found ininfective Dengue virus particle envelopes because the envelope is hostcell derived. The antibody of the present invention may substantiallyincrease the recognition of Dengue virus-infected cells by binding tothe surface of Dengue virus-infected cell. Antibody bound to the surfaceof infected cells may facilitate antibody-dependent cellularcytotoxicity (ADCC) or cell dependent cytotoxicity (CDC) and therebyenhance the killing of virus-infected cells and interrupting the virusreplication cycle. The interruption of the virus replication cycle mayreduce the severity of infection and speed recovery from disease. Thecapacity of an anti-Dengue virus antibody to inhibit Dengue virus NSprotein-dependent activity may be assessed by at least one suitableDengue virus NS protein assay, an infectivity assay or a replicationcycle assay, as described herein and/or as known in the art. Ananti-Dengue virus antibody of the invention can be of any class (IgG,IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambdalight chain. In one embodiment, the antibody comprises an IgG heavychain or defined fragment, for example, at least one of isotypes IgG1,IgG2, IgG3 or IgG4. Antibodies of this type can be prepared by employinga transgenic mouse or other trangenic non-human mammal comprising atleast one human light chain (e.g., IgG, IgA and IgM (e.g., γ1, γ2, γ3,γ4) transgenes as described herein and/or as known in the art. Inanother embodiment, the anti-Dengue virus antibody comprises an IgG1heavy chain and a IgG1 light chain.

[0099] At least one antibody of the invention binds at least onespecified epitope specific to at least one Dengue virus protein,subunit, fragment, portion or any combination thereof. At least oneepitope can comprise at least one antibody binding region that comprisesat least one portion of said protein, which epitope is preferablycomprised of at least one extracellular, structural, non-structural,soluble, hydrophilic, external or cytoplasmic portion of said protein.The at least one specified epitope can comprise any combination of atleast one amino acid sequence of at least one to three amino acids, tothe entire specified portion of contiguous amino acids of the Denguevirus protein.

[0100] Generally, the human antibody or antigen-binding fragment of thepresent invention will comprise an antigen-binding region that comprisesat least one human complementarity determining region (CDR1, CDR2 andCDR3) or variant of at least one heavy chain variable region and atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one light chain variable region. As anon-limiting example, the antibody or antigen-binding portion or variantcan comprise at least one of the heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 3, and/or a light chain CDR3 having the aminoacid sequence of SEQ ID NO: 4. In a particular embodiment, the antibodyor antigen-binding fragment can have an antigen-binding region thatcomprises at least a portion of at least one heavy chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NO: 3). In anotherparticular embodiment, the antibody or antigen-binding portion orvariant can have an antigen-binding region that comprises at least aportion of at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NO: 4). In a preferred embodiment the three heavy chainCDRs and the three light chain CDRs of the antibody or antigen-bindingfragment have the amino acid sequence of the corresponding CDR of atleast one of mAb (such as the anti-dengue virus antibody referred toherein as DEN3, which is not to be confused with the Dengue virusserotype 3 known as DEN-3), as described herein. Such antibodies can beprepared by chemically joining together the various portions (e.g.,CDRs, framework) of the antibody using conventional techniques, bypreparing and expressing a (i.e., one or more) nucleic acid moleculethat encodes the antibody using conventional techniques of recombinantDNA technology or by using any other suitable method.

[0101] The anti-Dengue virus antibody can comprise at least one of aheavy or light chain variable region having a defined amino acidsequence. For example, in a preferred embodiment, the anti-Dengue virusantibody comprises at least one of at least one heavy chain variableregion, optionally having the amino acid sequence of SEQ ID NO: 3 and/orat least one light chain variable region, optionally having the aminoacid sequence of SEQ ID NO: 4. Antibodies that bind to Dengue virus andthat comprise a defined heavy or light chain variable region can beprepared using suitable methods, such as phage display (Katsube et al.,1(5) INT. J. MOL. MED., 863-868 (1998)) or methods that employtransgenic animals, as known in the art and/or as described herein. Forexample, a transgenic mouse, comprising a functionally rearranged humanimmunoglobulin heavy chain transgene and a transgene comprising DNA froma human immunoglobulin light chain locus that can undergo functionalrearrangement, can be immunized with Dengue virus or a fragment thereofto elicit the production of antibodies. If desired, the antibodyproducing cells can be isolated and hybridomas or other immortalizedantibody-producing cells can be prepared as described herein and/or asknown in the art. Alternatively, the antibody, specified portion orvariant can be expressed using the encoding nucleic acid or portionthereof in a suitable host cell.

[0102] The invention also relates to antibodies, antigen-bindingfragments, immunoglobulin chains and CDRs comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Preferably, such antibodies or antigen-bindingfragments and antibodies comprising such chains or CDRs can bind Denguevirus with high affinity (e.g., K_(D) less than or equal to about 10⁻⁹M). Amino acid sequences that are substantially the same as thesequences described herein include sequences comprising conservativeamino acid substitutions, as well as amino acid deletions and/orinsertions. A conservative amino acid substitution refers to thereplacement of a first amino acid by a second amino acid that haschemical and/or physical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T. Amino Acid Codes

[0103] The amino acids that make up anti-Dengue virus antibodies of thepresent invention are often abbreviated. The amino acid designations canbe indicated by designating the amino acid by its single letter code,its three letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts et al., Molecular Biology of The Cell3^(rd) Edition, (Garland Publishing, Inc., New York, (1994)): SingleThree Letter Code Letter Code Name Three Nucleotide Codon(S) A AlaAlanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Aspartic acidGAG, GAU E Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU G GlyGlycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile IsoleucineAUA, AUC, AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC,CUG, CUU M Met Methionine AUG N Asn Asparagine AAC, AAU P Pro ProlineCCA, CCC, CCG, CCU Q Gln Glutamine CAA, GAG R Arg Arginine AGA, AGG,CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T ThrThreonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W TrpTryptophan UGG Y Tyr Tyrosine UAC, UAU

[0104] An anti-Dengue virus antibody of the present invention caninclude one or more amino acid substitutions, deletions or additions,either from natural mutations or human manipulation, as specifiedherein.

[0105] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of amino acid substitutions,insertions or deletions for any given anti-Dengue virus antibody,fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1 to 30 orany range or value therein, as specified herein.

[0106] Amino acids in an anti-Dengue virus antibody of the presentinvention that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,244 SCIENCE, 1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to, at least one Dengue virus NS protein binding activity. Sitesthat are critical for antibody binding can also be identified bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., 224 J. MOL. BIOL., 899-904(1992), and de Vos et al., 255 SCIENCE, 306-312 (1992)).

[0107] Anti-Dengue virus antibodies of the present invention caninclude, but are not limited to, at least one portion, sequence orcombination selected from five to all of the contiguous amino acids ofat least one of SEQ ID NOS: 3 and 4.

[0108] A(n) anti-Dengue virus antibody can further optionally comprise apolypeptide of at least one of 70% to 100% of the contiguous amino acidsof at least one of SEQ ID NOS: 3 and 4.

[0109] In one embodiment, the amino acid sequence of an immunoglobulinchain, or portion thereof (e.g., variable region, CDR) has about 70% to100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or any range or value therein) to the amino acid sequence of thecorresponding chain of SEQ ID NO: 4. For example, the amino acidsequence of a light chain variable region can be compared with thesequence of SEQ ID NO: 4, or the amino acid sequence of a heavy chainCDR3 can be compared with SEQ ID NO: 3. Preferably, 70% to 100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

[0110] Exemplary heavy chain and light chain variable regions sequencesare provided in SEQ ID NOS: 3 and 4. The antibodies of the presentinvention, or specified variants thereof, can comprise any number ofcontiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10% to 100% of the number of contiguous residues inan anti-Dengue virus antibody. Optionally, this subsequence ofcontiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250 or more amino acids in length, or any range or valuetherein. Further, the number of such subsequences can be any integerselected from the group consisting of from 1 to 20, such as at least 2,3, 4 or 5.

[0111] In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

[0112] The modified antibodies and antigen-binding fragments of theinvention can comprise one or more organic moieties that are covalentlybonded, directly or indirectly, to the antibody. Each organic moietythat is bonded to an antibody or antigen-binding fragment of theinvention can independently be a hydrophilic polymeric group, a fattyacid group or a fatty acid ester group. As used herein, the term “fattyacid” encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

[0113] Fatty acids and fatty acid esters suitable for modifyingantibodies of the invention can be saturated or can contain one or moreunits of unsaturation. Fatty acids that are suitable for modifyingantibodies of the invention include, for example, n-dodecanoate (C₁₂,laurate), n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈,stearate), n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂,behenate), n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀),cis-Δ9-octadecanoate (C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate(C₂₀, arachidonate), octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like. Suitable fattyacid esters include monoesters of dicarboxylic acids that comprise alinear or branched lower alkyl group. The lower alkyl group can comprisefrom one to about twelve, preferably one to about six, carbon atoms.

[0114] The modified antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, G. T. Hermanson, Bioconiugate Techniques,(Academic Press, San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C1-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221, the entire teachings of which areincorporated herein by reference.)

[0115] The modified antibodies of the invention can be produced byreacting an antibody or antigen-binding fragment with a modifying agent.For example, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified antibodies or antigen-bindingfragments can also be prepared by reducing disulfide bonds (e.g.,intra-chain disulfide bonds) of an antibody or antigen-binding fragment.The reduced antibody or antigen-binding fragment can then be reactedwith a thiol-reactive modifying agent to produce the modified antibodyof the invention. Modified antibodies and antigen-binding fragmentscomprising an organic moiety that is bonded to specific sites of anantibody of the present invention can be prepared using suitablemethods, such as reverse proteolysis (Fisch et al., 3 BIOCONJUGATECHEM., 147-153 (1992); Werlen et al., 5 BIOCONJUGATE CHEM., 411-417(1994); Kumaran et al., 6(10) PROTEIN SCI., 2233-2241 (1997); Itoh etal., 24(1) BIOORG. CHEM., 59-68 (1996); Capellas et al., 56(4)BIOTECHNOL. BIOENG., 456-463 (1997)), and the methods described in G. T.Hermanson, Bioconjugate Techniques, (Academic Press, San Diego, Calif.(1996)).

[0116] Anti-Dengue Virus Antibody Compositions

[0117] The present invention also provides at least one anti-Denguevirus antibody composition comprising at least one, at least two, atleast three, at least four, at least five, at least six or moreanti-Dengue virus antibodies thereof, as described herein and/or asknown in the art that are provided in a non-naturally occurringcomposition, mixture or form. Such compositions comprise non-naturallyoccurring compositions comprising at least one or two full length, C-and/or N-terminally deleted variants, domains, fragments, or specifiedvariants, of the anti-Dengue virus antibody amino acid sequence selectedfrom the group consisting of 70% to 100% of the contiguous amino acidsof SEQ ID NOS: 3 and 4 or specified fragments, domains or variantsthereof. Preferred anti-Dengue virus antibody compositions include atleast one or two full length, fragments, domains or variants as at leastone CDR or LBR containing portions of the anti-Dengue virus antibodysequence of 70% to 100% of SEQ ID NOS: 3 or 4 or specified fragments,domains or variants thereof. Further preferred compositions comprise 40%to 99% of at least one of 70% to 100% of SEQ ID NOS: 3 or 4 or specifiedfragments, domains or variants thereof. Such composition percentages areby weight, volume, concentration, molarity, or molality as liquid or drysolutions, mixtures, suspension, emulsions or colloids, as known in theart or as described herein.

[0118] Anti-Dengue virus antibody compositions of the present inventioncan further comprise at least one of any suitable and effective amountof a composition or pharmaceutical composition comprising at least oneanti-Dengue virus antibody to a cell, tissue, organ, animal or patientin need of such modulation, treatment or therapy, optionally furthercomprising at least one selected from at least one Dengue virusanti-viral agent (e.g., but not limited to, an anti-Dengue virusantibody or fragment, a soluble Dengue virus receptor or fragment,fusion proteins thereof, or a small molecule Dengue virus infectivity orreplication antagonist), an anti-rheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a flurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyme), a growth factorcytokine or a growth factor cytokine antagonist. Non-limiting examplesof such growth factors or cytokines include, but are not limited to, anyof Erythropoietin, interferons, GCSF, GMCSF, and IL-1 to IL-23. Suitabledosages are well known in the art. See, e.g., Wells et al. (Eds.),Pharmacotherapy Handbook, 2^(nd) Edition, (Appleton and Lange, Stamford,Conn. (2000)); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000,Deluxe Edition, (Tarascon Publishing, Loma Linda, Calif. (2000)), eachof which references are entirely incorporated herein by reference.

[0119] Such anti-cancer, anti-viral or anti-infectives can also includetoxin molecules that are associated, bound, co-formulated orco-administered with at least one antibody of the present invention. Thetoxin can optionally act to selectively kill the pathologic cell ortissue. The pathologic cell can be a cancer or other cell. Such toxinscan be, but are not limited to, purified or recombinant toxin or toxinfragment comprising at least one functional cytotoxic domain of toxin,e.g., selected from at least one of ricin, diphtheria toxin, a venomtoxin, or a bacterial toxin. The term toxin also includes bothendotoxins and exotoxins produced by any naturally occurring, mutant orrecombinant bacteria or viruses which may cause any pathologicalcondition in humans and other mammals, including toxin shock, which canresult in death. Such toxins may include, but are not limited to,enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stableenterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxicshock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B(SEB), or C (SEC), Streptococcal enterotoxins and the like. Suchbacteria include, but are not limited to, strains of a species ofenterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., strainsof serotype 0157:H7), Staphylococcus species (e.g., Staphylococcusaureus, Staphylococcus pyogenes), Shigella species (e.g., Shigelladysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei),Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis,Salmonella enteritidis), Clostridium species (e.g., Clostridiumperfringens, Clostridium dificile, Clostridium botulinum), Camphlobacterspecies (e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacterspecies, (e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonassobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonasshigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrioscholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonasaeruginosa, and Streptococci. See, e.g., Stein, ed., Internal Medicine,3^(rd) Edition, pp 1-13, (Little, Brown and Co., Boston, Mass. (1990));Evans et al. (Eds.), Bacterial Infections of Humans: Epidemiology andControl, 2^(nd) Edition, pp 239-254, (Plenum Medical Book Co., New York,N.Y. (1991)); Mandell et al, Principles and Practice of InfectiousDiseases, 3^(rd) Edition, (Churchill Livingstone, New York, N.Y.(1990)); Berkow et al. (Eds.), The Merck Manual, 16^(th) Edition, (Merckand Co., Rahway, N.J., (1992)); Wood et al, 76 FEMS MICROBIOLOGYIMMUNOLOGY, 121-134 (1991); Marrack et al, 248 SCIENCE, 705-711 (1990),the contents of which references are incorporated entirely herein byreference.

[0120] Anti-Dengue virus antibody compounds, compositions orcombinations of the present invention can further comprise at least oneof any suitable auxiliary, such as, but not limited to, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvantor the like. Pharmaceutically acceptable auxiliaries are preferred.Non-limiting examples of, and methods of preparing such sterilesolutions are well known in the art, such as, but limited to, Gennaro(Ed.), Remington's Pharmaceutical Sciences 18^(th) Edition, (MackPublishing Co., Easton, Pa. (1990)). Pharmaceutically acceptablecarriers can be routinely selected that are suitable for the mode ofadministration, solubility and/or stability of the anti-Dengue virusantibody, fragment or variant composition as well known in the art or asdescribed herein.

[0121] Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1% to 99.99% byweight or volume. Exemplary protein excipients include serum albuminsuch as human serum albumin (HSA), recombinant human albumin (rHA),gelatin, casein, and the like. Representative amino acid/antibodycomponents, which can also function in a buffering capacity, includealanine, glycine, arginine, betaine, histidine, glutamic acid, asparticacid, cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like.

[0122] Carbohydrate excipients suitable for use in the inventioninclude, for example, monosaccharides such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and thelike. Preferred carbohydrate excipients for use in the present inventionare mannitol, trehalose, and raffinose.

[0123] Anti-Dengue virus antibody compositions can also include a bufferor a pH-adjusting agent; typically, the buffer is a salt prepared froman organic acid or base. Representative buffers include organic acidsalts such as salts of citric acid, ascorbic acid, gluconic acid,carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalicacid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferredbuffers for use in the present compositions are organic acid salts suchas citrate.

[0124] Additionally, anti-Dengue virus antibody compositions of theinvention can include polymeric excipients/additives such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethyleneglycols, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids),steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0125] These and additional known pharmaceutical excipients and/oradditives suitable for use in the anti-Dengue virus antibody, portion orvariant compositions according to the invention are known in the art,e.g., as listed in Remington: The Science & Practice of Pharmacy,19^(th) Edition, (Williams & Williams, (1995)), and in the Physician'sDesk Reference, 52^(nd) Edition, (Medical Economics, Montvale, N.J.(1998)), the disclosures of which are entirely incorporated herein byreference. Preferred carrier or excipient materials are carbohydrates(e.g., saccharides and alditols) and buffers (e.g., citrate) orpolymeric agents.

[0126] Formulations

[0127] As noted above, the invention provides for stable formulations,which is preferably a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservativeas well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one anti-Dengue virus antibody ina pharmaceutically acceptable formulation. Preserved formulationscontain at least one known preservative or optionally selected from thegroup consisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001% to 5%, or anyrange or value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1% to2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1% to 3% benzylalcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001% to 0.5%thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25,0.28, 0.5, 0.9, 1.0%), 0.0005% to 1.0% alkylparaben(s) (e.g., 0.00075,0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075,0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0128] As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-Dengue virus antibody with the prescribedbuffers and/or preservatives, optionally in an aqueous diluent, whereinsaid packaging material comprises a label that indicates that suchsolution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20,24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater. The inventionfurther comprises an article of manufacture, comprising packagingmaterial, a first vial comprising lyophilized at least one anti-Denguevirus antibody, and a second vial comprising an aqueous diluent ofprescribed buffer or preservative, wherein said packaging materialcomprises a label that instructs a patient to reconstitute the at leastone anti-Dengue virus antibody in the aqueous diluent to form a solutionthat can be held over a period of twenty-four hours or greater.

[0129] The at least one anti-Dengue virus antibody used in accordancewith the present invention can be produced by recombinant means,including from mammalian cell or transgenic preparations, or can bepurified from other biological sources, as described herein or as knownin the art.

[0130] The range of at least one anti-Dengue virus antibody in theproduct of the present invention includes amounts yielding uponreconstitution, if in a wet/dry system, concentrations from about 1.0μg/ml to about 1000 mg/ml, although lower and higher concentrations areoperable and are dependent on the intended delivery vehicle, e.g.,solution formulations will differ from transdermal patch, pulmonary,transmucosal, or osmotic or micro pump methods.

[0131] Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

[0132] Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

[0133] Other additives, such as a pharmaceutically acceptablesolubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate),Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80(polyoxyethylene (20) sorbitan monooleate), Pluronic F68(polyoxyethylene polyoxypropylene block copolymers), and PEG(polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers,and chelators such as EDTA and EGTA can optionally be added to theformulations or compositions to reduce aggregation. These additives areparticularly useful if a pump or plastic container is used to administerthe formulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

[0134] The formulations of the present invention can be prepared by aprocess which comprises mixing at least one anti-Dengue virus antibodyand a preservative selected from the group consisting of phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal ormixtures thereof in an aqueous diluent. Mixing the at least oneanti-Dengue virus antibody and preservative in an aqueous diluent iscarried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one anti-Dengue virus antibody in buffered solution is combinedwith the desired preservative in a buffered solution in quantitiessufficient to provide the protein and preservative at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that can be optimizedfor the concentration and means of administration used.

[0135] The claimed formulations can be provided to patients as clearsolutions or as dual vials comprising a vial of lyophilized at least oneanti-Dengue virus antibody that is reconstituted with a second vialcontaining water, a preservative and/or excipients, preferably aphosphate buffer and/or saline and a chosen salt, in an aqueous diluent.Either a single solution vial or dual vial requiring reconstitution canbe reused multiple times and can suffice for a single or multiple cyclesof patient treatment and thus can provide a more convenient treatmentregimen than currently available.

[0136] The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2° C. to about 40° C. and retain the biologically activity of theprotein for extended periods of time, thus, allowing a package labelindicating that the solution can be held and/or used over a period of 6,12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent isused, such label can include use up to one to twelve months, one-half,one and a half, and/or two years.

[0137] The solutions of at least one anti-Dengue virus antibody in theinvention can be prepared by a process that comprises mixing at leastone antibody in an aqueous diluent. Mixing is carried out usingconventional dissolution and mixing procedures. To prepare a suitablediluent, for example, a measured amount of at least one antibody inwater or buffer is combined in quantities sufficient to provide theprotein and optionally a preservative or buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that can be optimizedfor the concentration and means of administration used.

[0138] The claimed products can be provided to patients as clearsolutions or as dual vials comprising a vial of lyophilized at least oneanti-Dengue virus antibody that is reconstituted with a second vialcontaining the aqueous diluent. Either a single solution vial or dualvial requiring reconstitution can be reused multiple times and cansuffice for a single or multiple cycles of patient treatment and thusprovides a more convenient treatment regimen than currently available.

[0139] The claimed products can be provided indirectly to patients byproviding to pharmacies, clinics, or other such institutions andfacilities, clear solutions or dual vials comprising a vial oflyophilized at least one anti-Dengue virus antibody that isreconstituted with a second vial containing the aqueous diluent. Theclear solution in this case can be up to one liter or even larger insize, providing a large reservoir from which smaller portions of the atleast one antibody solution can be retrieved one or multiple times fortransfer into smaller vials and provided by the pharmacy or clinic totheir customers and/or patients.

[0140] Recognized devices comprising these single vial systems includethose pen-injector devices for delivery of a solution such as BD Pens,BD Autojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject (Portland, Oreg., www.bioiect.com); NationalMedical Products, Weston Medical (Peterborough, United Kingdom,www.weston-medical.com), and Medi-Ject Corporation (Minneapolis, Minn.,www.mediiect.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

[0141] The products presently claimed include packaging material. Thepackaging material provides, in addition to the information required bythe regulatory agencies, the conditions under which the product can beused. The packaging material of the present invention providesinstructions to the patient to reconstitute the at least one anti-Denguevirus antibody in the aqueous diluent to form a solution and to use thesolution over a period of two to twenty-four hours or greater for thetwo vial, wet/dry, product. For the single vial, solution product, thelabel indicates that such solution can be used over a period of two totwenty-four hours or greater. The presently claimed products are usefulfor human pharmaceutical product use.

[0142] The formulations of the present invention can be prepared by aprocess that comprises mixing at least one anti-Dengue virus antibodyand a selected buffer, preferably a phosphate buffer containing salineor a chosen salt. Mixing the at least one antibody and buffer in anaqueous diluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

[0143] The claimed stable or preserved formulations can be provided topatients as clear solutions or as dual vials comprising a vial oflyophilized at least one anti-Dengue virus antibody that isreconstituted with a second vial containing a preservative or buffer andexcipients in an aqueous diluent. Either a single solution vial or dualvial requiring reconstitution can be reused multiple times and cansuffice for a single or multiple cycles of patient treatment and thusprovides a more convenient treatment regimen than currently available.

[0144] At least one anti-Dengue virus antibody in either the stable orpreserved formulations or solutions described herein, can beadministered to a patient in accordance with the present invention via avariety of delivery methods including SC or IM injection; transdermal,pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump,or other means appreciated by the skilled artisan, as well-known in theart.

[0145] Therapeutic Applications

[0146] The present invention also provides a method for modulating ortreating at least one Dengue virus related disease or condition, in acell, tissue, organ, animal, or patient, as known in the art or asdescribed herein, using at least one anti-Dengue virus antibody of thepresent invention. The present invention also provides methods formodulating, treating or preventing Dengue virus infection in a subject.The present invention also provides methods for modulating, treating,inhibiting or blocking Dengue virus infectivity or replication in acell, tissue, organ, animal or patient.

[0147] Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-Dengue virus antibody to a cell, tissue,organ, animal or patient in need of such modulation, treatment,prevention or therapy. Such a method can optionally further compriseco-administration or combination therapy for such modulation, treatment,prevention or therapy, wherein the administering of said at least oneanti-Dengue virus antibody, specified portion or variant thereof,further comprises administering, before, concurrently, and/or after, atleast one agent selected from at least one Dengue virus antagonist(e.g., but not limited to, a Dengue virus antibody or fragment, asoluble Dengue virus receptor or fragment, fusion proteins thereof, or asmall molecule Dengue virus antagonist), an anti-rheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an anti-infective agent, ananti-tumor agent, an anti-proliferative agent, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al. (Eds.), Pharmacotherapy Handbook. 2^(nd) Edition,(Appleton and Lange, Stamford, Conn. (2000)); and PDR Pharmacopoeia,Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, (TarasconPublishing, Loma Linda, Calif. (2000)), each of which references areentirely incorporated herein by reference.

[0148] As used herein, an “Anti-Dengue virus antibody” or fragment andthe like decreases, blocks, inhibits, abrogates or interferes withDengue virus activity infectivity or replication, in vitro, in situand/or preferably in vivo. For example, a suitable Dengue virus antibodyof the present invention can bind Dengue virus or Dengue virus-infectedcells and includes whole anti-Dengue virus antibodies, antigen-bindingfragments thereof, and specified mutants or domains thereof that bindspecifically to Dengue virus or Dengue virus infected cells. A suitableDengue virus antibody or fragment can also decrease, block, abrogate,interfere, prevent and/or inhibit Dengue virus RNA, DNA or proteinsynthesis, Dengue virus release, Dengue virus cell receptor interaction,membrane Dengue virus cleavage, Dengue virus activity, Dengue virusreplication, production and/or synthesis.

[0149] Preferred methods for determining monoclonal antibody specificityand affinity by competitive inhibition can be found in Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988)); Colligan et al. (Eds.), CurrentProtocols in Immunology, (Greene Publishing Assoc. and WileyInterscience, New York, (1992-2000)); Kozbor et al., 4 IMMUNOL. TODAY,72-79 (1983); Ausubel et al. (Eds.), Current Protocols in MolecularBiology, (Wiley Interscience, New York (1987-2000)); and Muller, 92METH. ENZYMOL., 589-601 (1983)), which references are entirelyincorporated herein by reference.

[0150] Cytokines include any known cytokine. See, e.g.,www.CopewithCytokines.com. Cytokine antagonists include, but are notlimited to, any antibody, fragment or mimetic, any soluble receptor,fragment or mimetic, any small molecule antagonist, or any combinationthereof.

[0151] Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-Denguevirus antibody composition that total, on average, a range from at leastabout 0.01 to 500 milligrams of at least one anti-Dengue virus antibodyper kilogram of patient per dose, and preferably from at least about 0.1to 100 milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1 to 5000 μg/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

[0152] Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 to 500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

[0153] Alternatively, the dosage administered can vary depending uponknown factors, such as the pharmacodynamic characteristics of theparticular agent, and its mode and route of administration; age, health,and weight of the recipient; nature and extent of symptoms, kind ofconcurrent treatment, frequency of treatment, and the effect desired.Usually a dosage of active ingredient can be about 0.1 to 100 milligramsper kilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

[0154] As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

[0155] Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5 to 99.999% by weight based on the total weight ofthe composition.

[0156] For parenteral administration, the antibody can be formulated asa solution, suspension, emulsion or lyophilized powder in association,or separately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1% to 10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

[0157] Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

[0158] Parenteral Formulations and Administration

[0159] Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemi-synthetic fatty oils or fatty acids; natural or synthetic orsemi-synthetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

[0160] Delivery

[0161] The invention further relates to the administration of at leastone anti-Dengue virus antibody by parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermalmeans. At least one anti-Dengue virus antibody composition can beprepared for use for parenteral (subcutaneous, intramuscular orintravenous) or any other administration particularly in the form ofliquid solutions or suspensions; for use in vaginal or rectaladministration particularly in semisolid forms such as, but not limitedto, creams and suppositories; for buccal, or sublingual administrationsuch as, but not limited to, in the form of tablets or capsules; orintranasally such as, but not limited to, the form of powders, nasaldrops or aerosols or certain agents; or transdermally such as notlimited to a gel, ointment, lotion, suspension or patch delivery systemwith chemical enhancers such as dimethyl sulfoxide to either modify theskin structure or to increase the drug concentration in the transdermalpatch (Junginger et al., Drug Permeation Enhancement, pp. 59-90 (D. S.Hsieh (Ed.), Marcel Dekker, Inc., New York (1994)), entirelyincorporated herein by reference), or with oxidizing agents that enablethe application of formulations containing proteins and peptides ontothe skin (WO 98/53847), or applications of electric fields to createtransient transport pathways such as electroporation, or to increase themobility of charged drugs through the skin such as iontophoresis, orapplication of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989and 4,767,402) (the above publications and patents being entirelyincorporated herein by reference).

[0162] Pulmonary/Nasal Administration

[0163] For pulmonary administration, preferably at least one anti-Denguevirus antibody composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses. According to theinvention, at least one anti-Dengue virus antibody can be delivered byany of a variety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of antibodies are alsoknown in the art. All such devices can use of formulations suitable forthe administration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellant gas and requireactuation during inspiration (See, e.g., International Publication Nos.WO 94/16970 and WO 98/35888). Dry powder inhalers like Turbuhaler™(Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura),devices marketed by Inhale Therapeutics, and the Spinhaler® powderinhaler (Fisons), use breath-actuation of a mixed powder (U.S. Pat. No.4,668,218, Astra; EP 237507, Astra; International Publication No. WO97/25086, Glaxo; International Publication No. WO 94/08552, Dura; U.S.Pat. No. 5,458,135, Inhale; and International Publication No. WO94/06498, Fisons, entirely incorporated herein by reference). Nebulizerslike AERx™ Aradigm, the Ultravent® nebulizer (Mallinckrodt), and theAcorn II® nebulizer (Marquest Medical Products) (U.S. Pat. No.5,404,871, Aradigm; and International Publication No. WO 97/22376), theabove references entirely incorporated herein by reference, produceaerosols from solutions, while metered dose inhalers, dry powderinhalers, etc. generate small particle aerosols. These specific examplesof commercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-Dengue virusantibody is delivered by a dry powder inhaler or a sprayer. There are aseveral desirable features of an inhalation device for administering atleast one antibody of the present invention. For example, delivery bythe inhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 μm, preferably about 1 to 5 μm, forgood respirability.

[0164] Administration of Anti-Dengue Virus Antibody Compositions as aSpray

[0165] A spray including Dengue virus antibody composition protein canbe produced by forcing a suspension or solution of at least oneanti-Dengue virus antibody through a nozzle under pressure. The nozzlesize and configuration, the applied pressure, and the liquid feed ratecan be chosen to achieve the desired output and particle size. Anelectrospray can be produced, for example, by an electric field inconnection with a capillary or nozzle feed. Advantageously, particles ofat least one anti-Dengue virus antibody composition protein delivered bya sprayer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

[0166] Formulations of at least one anti-Dengue virus antibodycomposition protein suitable for use with a sprayer typically includeantibody composition protein in an aqueous solution at a concentrationof about 0.1 mg to about 100 mg of at least one anti-Dengue virusantibody composition protein per ml of solution or mg/gm, or any rangeor value therein, e.g., but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50,60, 70, 80, 90 or 100 mg/ml or mg/gm. The formulation can include agentssuch as an excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the antibody compositionprotein, such as a buffer, a reducing agent, a bulk protein, or acarbohydrate. Bulk proteins useful in formulating antibody compositionproteins include albumin, protamine, or the like. Typical carbohydratesuseful in formulating antibody composition proteins include sucrose,mannitol, lactose, trehalose, glucose, or the like. The antibodycomposition protein formulation can also include a surfactant, which canreduce or prevent surface-induced aggregation of the antibodycomposition protein caused by atomization of the solution in forming anaerosol. Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitol fatty acid esters. Amounts will generally range between 0.001%and 14% by weight of the formulation. Especially preferred surfactantsfor purposes of this invention are polyoxyethylene sorbitan monooleate,polysorbate 80, polysorbate 20, or the like. Additional agents known inthe art for formulation of a protein such as anti-Dengue virusantibodies, or specified portions or variants, can also be included inthe formulation.

[0167] Administration of Dengue Virus Antibody Compositions by aNebulizer

[0168] Antibody composition protein can be administered by a nebulizer,such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jetnebulizer, a compressed air source is used to create a high-velocity airjet through an orifice. As the gas expands beyond the nozzle, alow-pressure region is created, which draws a solution of antibodycomposition protein through a capillary tube connected to a liquidreservoir. The liquid stream from the capillary tube is sheared intounstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of antibody composition protein either directly or through acoupling fluid, creating an aerosol including the antibody compositionprotein. Advantageously, particles of antibody composition proteindelivered by a nebulizer have a particle size less than about 10 μm,preferably in the range of about 1 μm to about 5 μm, and most preferablyabout 2 μm to about 3 μm.

[0169] Formulations of at least one anti-Dengue virus antibody suitablefor use with a nebulizer, either jet or ultrasonic, typically include aconcentration of about 0.1 mg to about 100 mg of at least oneanti-Dengue virus antibody protein per ml of solution. The formulationcan include agents such as an excipient, a buffer, an isotonicity agent,a preservative, a surfactant, and, preferably, zinc. The formulation canalso include an excipient or agent for stabilization of the at least oneanti-Dengue virus antibody composition protein, such as a buffer, areducing agent, a bulk protein, or a carbohydrate. Bulk proteins usefulin formulating at least one anti-Dengue virus antibody compositionproteins include albumin, protamine, or the like. Typical carbohydratesuseful in formulating at least one anti-Dengue virus antibody includesucrose, mannitol, lactose, trehalose, glucose, or the like. The atleast one anti-Dengue virus antibody formulation can also include asurfactant, which can reduce or prevent surface-induced aggregation ofthe at least one anti-Dengue virus antibody caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbital fatty acid esters. Amounts will generally rangebetween 0.001 and 4% by weight of the formulation. Especially preferredsurfactants for purposes of this invention are polyoxyethylene sorbitanmono-oleate, polysorbate 80, polysorbate 20, or the like. Additionalagents known in the art for formulation of a protein such as antibodyprotein can also be included in the formulation.

[0170] Administration of Dengue Virus Antibody Compositions by a MeteredDose Inhaler

[0171] In a metered dose inhaler (MDI), a propellant, at least oneanti-Dengue virus antibody, and any excipients or other additives arecontained in a canister as a mixture including a liquefied compressedgas. Actuation of the metering valve releases the mixture as an aerosol,preferably containing particles in the size range of less than about 10μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μmto about 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

[0172] Formulations of at least one anti-Dengue virus antibody for usewith a metered-dose inhaler device will generally include a finelydivided powder containing at least one anti-Dengue virus antibody as asuspension in a non-aqueous medium, for example, suspended in apropellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-Dengue virus antibody as asuspension in the propellant, to protect the active agent againstchemical degradation, and the like. Suitable surfactants includesorbitan trioleate, soya lecithin, oleic acid, or the like. In somecases solution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein such asprotein can also be included in the formulation.

[0173] One of ordinary skill in the art will recognize that the methodsof the current invention can be achieved by pulmonary administration ofat least one anti-Dengue virus antibody compositions via devices notdescribed herein.

[0174] Oral Formulations and Administration

[0175] Formulations for oral administration may rely on theco-administration of adjuvants (e.g., resorcinols and nonionicsurfactants such as polyoxyethylene oleyl ether andn-hexadecylpolyethylene ether) to increase artificially the permeabilityof the intestinal walls, as well as the co-administration of enzymaticinhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semi-synthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, α-tocopherol, antioxidant such ascysteine, disintegrator, binder, thickener, buffering agent, sweeteningagent, flavoring agent, perfuming agent, etc.

[0176] Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. Nos. 5,879,681 and 5,871,753 are usedto deliver biologically active agents orally are known in the art.

[0177] Mucosal Formulations and Administration

[0178] For absorption through mucosal surfaces, compositions and methodsof administering at least one anti-Dengue virus antibody include anemulsion comprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucoussurfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

[0179] Transdermal Formulations and Administration

[0180] For transdermal administration, the at least one anti-Denguevirus antibody is encapsulated in a delivery device such as a liposomeor polymeric nanoparticles, microparticle, microcapsule, or microspheres(referred to collectively as microparticles unless otherwise stated). Anumber of suitable devices are known, including microparticles made ofsynthetic polymers such as polyhydroxy acids such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

[0181] Prolonged Administration and Formulations

[0182] It can be sometimes desirable to deliver the compounds of thepresent invention to the subject over prolonged periods of time, forexample, for periods of one week to one year from a singleadministration. Various slow release, depot or implant dosage forms canbe utilized. For example, a dosage form can contain a pharmaceuticallyacceptable non-toxic salt of the compounds that has a low degree ofsolubility in body fluids, for example, (a) an acid addition salt with apolybasic acid such as phosphoric acid, sulfuric acid, citric acid,tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamicacid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, andthe like; (b) a salt with a polyvalent metal cation such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium and the like, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and Sustained and Controlled Release Drug Delivery Systems, (J. R.Robinson (Ed.), Marcel Dekker, Inc., New York (1978)).

[0183] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLE 1 Cloning and Expression of Dengue Virus Antibody in MammalianCells

[0184] A typical mammalian expression vector contains at least onepromoter element, which mediates the initiation of transcription ofmRNA, the antibody coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription can be achieved with the early and latepromoters from SV40, the long terminal repeats (LTRS) from Retroviruses,e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus(CMV). However, cellular elements can also be used (e.g., the humanactin promoter). Suitable expression vectors for use in practicing thepresent invention include, for example, vectors such as pIRES1neo,pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto,Calif.), pcDNA3.1 (+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−)(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian hostcells that could be used include human Hela 293, H9 and Jurkat cells,mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells,mouse L cells and Chinese hamster ovary (CHO) cells.

[0185] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells. [0163]The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., 227 BIOCHEM. J.,277-279 (1991); and Bebbington et al., 10 BIO/TECHNOLOGY, 169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

[0186] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., 5 MOLEC. CELL. BIOL.,438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart et al., 41CELL, 521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

[0187] Cloning and Expression in CHO Cells

[0188] The vector pC4 may be used for the expression of Dengue virusantibody. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCCAccession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary or other cellslacking dihydrofolate activity that are transfected with these plasmidscan be selected by growing the cells in a selective medium (e.g., alphaminus MEM, Life Technologies, Gaithersburg, Md.) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt et al., 253 J. BIOL. CHEM., 1357-1370 (1978); J. L. Hamlin andC. Ma, 1097 BIOCHEM. ET BIOPHYS. ACTA, 107-143 (1990); and M. J. Pageand M. A. Sydenham, 9 BIOTECHNOLOGY, 64-68 (1991)). Cells grown inincreasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach can be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained that contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

[0189] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen et al., 5 MOLEC. CELL. BIOL., 438-447 (1985)) plus afragment isolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart et al., 41 CELL, 521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the Denguevirus in a regulated way in mammalian cells (M. Gossen and H. Bujard, 89PROC. NATL. ACAD. SCI. USA, 5547-5551 (1992)). For the polyadenylationof the mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

[0190] The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

[0191] The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

[0192] Chinese hamster ovary (CHO) cells lacking an active DHFR gene areused for transfection. 5 μg of the expression plasmid pC4 isco-transfected with 0.5 μg of the plasmid pSV2-neo using lipofectin. Theplasmid pSV2neo contains a dominant selectable marker, the neo gene fromTn5 encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After two days, the cells are trypsinized and seededin hybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about ten to fourteen days, single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained that grow at a concentration of100-200 mM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

EXAMPLE 2

[0193] Generation of High Affinity Human IgG Monoclonal AntibodiesReactive with Human Dengue Virus Using Transgenic Mice

[0194] Transgenic mice may be used that contain human heavy and lightchain immunoglobulin genes to generate high affinity, completely human,monoclonal antibodies that can be used therapeutically to inhibit theaction of Dengue virus for the treatment of one or more Denguevirus-mediated disease. (CBA/J×C57/BL6/J) F₂ hybrid mice containinghuman variable and constant region antibody transgenes for both heavyand light chains are immunized with human recombinant Dengue virus(Taylor et al., 6 INTL. IMMUNOL., 579-591 (1993); Lonberg et al., 368NATURE, 856-859 (1994); M. Neuberger, 14 NATURE BIOTECH., 826 (1996);Fishwild et al., 14 NATURE BIOTECHNOLOGY, 845-851 (1996)). Severalfusions yielded one or more panels of completely human Dengue virusreactive IgG monoclonal antibodies. The completely human anti-Denguevirus antibodies are further characterized. Such antibodies are found tohave affinity constants somewhere between 1×10⁹ and 9×10¹². Theunexpectedly high affinities of these fully human monoclonal antibodiesmake them suitable candidates for therapeutic applications in Denguevirus related diseases, pathologies or disorders. Abbreviations BSAbovine serum albumin CO₂ carbon dioxide DMSO dimethyl sulfoxide EIAenzyme immunoassay FBS fetal bovine serum H₂O₂ hydrogen peroxide HRPhorseradish peroxidase ID Interadermal Ig Immunoglobulin IPIntraperitoneal IV Intravenous mAb monoclonal antibody OD opticaldensity OPD o-Phenylenediamine dihydrochloride PEG polyethylene glycolPSA penicillin, streptomycin, amphotericin RT room temperature SQSubcutaneous TNF┘ tissue necrosis factor alpha v/v volume per volume w/vweight per volume

[0195] Materials and Methods

[0196] Animals

[0197] Transgenic mice that can express human antibodies are known inthe art (and are commercially available (e.g., from Medarex San Jose,Calif.; Abgenix, Freemont, Calif., and others) that express humanimmunoglobulins, but not mouse IgM or Igκ. For example, such transgenicmice contain human sequence transgenes that undergo V(D)J joining,heavy-chain class switching, and somatic mutation to generate arepertoire of human sequence immunoglobulins (Lonberg et al., 368NATURE, 856-859 (1994)). The light chain transgene can be derived, e.g.,in part from a yeast artificial chromosome clone that includes nearlyhalf of the germline human Vκ region. In addition, the heavy-chaintransgene can encode both human μ and human γ1 (Fishwild et al., 14NATURE BIOTECHNOLOGY, 845-851 (1996)) and/or γ3 constant regions. Micederived from appropriate genotopic lineages can be used in theimmunization and fusion processes to generate fully human monoclonalantibodies to Dengue virus.

[0198] Immunization

[0199] One or more immunization schedules can be used to generate theanti-Dengue virus human hybridomas. The first several fusions can beperformed after the following exemplary immunization protocol, but othersimilar known protocols can be used. Several fourteen to twenty week-oldfemale and/or surgically castrated transgenic male mice are immunized IPand/or ID with 1 to 1000 μg of recombinant human Dengue virus emulsifiedwith an equal volume of TITERMAX or complete Freund's adjuvant in afinal volume of 100 to 400 μL (e.g., 200). Each mouse can alsooptionally receive 1 to 10 μg in 100 μL physiological saline at each oftwo SQ sites. The mice can then be immunized 1 to 7, 5 to 12, 10 to 18,17 to 25 and/or 21 to 34 days later IP (1 to 400 μg) and SQ (1 to 400μg×2) with Dengue virus emulsified with an equal volume of TITERMAX orincomplete Freund's adjuvant. Mice can be bled 12 to 25 and 25 to 40days later by retro-orbital puncture without anti-coagulant. The bloodis then allowed to clot at room temperature for one hour and the serumis collected and titered using an Dengue virus EIA assay according toknown methods. Fusions are performed when repeated injections do notcause titers to increase. At that time, the mice can be given a final IVbooster injection of 1 to 400 μg Dengue virus diluted in 100 μLphysiological saline. Three days later, the mice can be euthanized bycervical dislocation and the spleens removed aseptically and immersed in10 mL of cold phosphate buffered saline (PBS) containing 100 U/mLpenicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (PSA).The splenocytes are harvested by sterilely perfusing the spleen withPSA-PBS. The cells are washed once in cold PSA-PBS, counted using Trypanblue dye exclusion and re-suspended in RPMI 1640 media containing 25 mMHepes.

[0200] Cell Fusion

[0201] Fusion can be carried out at a 1:1 to 1:10 ratio of murinemyeloma cells to viable spleen cells according to known methods, e.g.,as known in the art. As a non-limiting example, spleen cells and myelomacells can be pelleted together. The pellet can then be slowlyre-suspended, over thirty seconds, in 1 mL of 50% (w/v) PEG/PBS solution(PEG molecular weight 1,450, Sigma) at 37° C. The fusion can then bestopped by slowly adding 10.5 mL of RPMI 1640 medium containing 25 mMHepes (37° C.) over one minute. The fused cells are centrifuged for fiveminutes at 500 to 1500 rpm. The cells are then re-suspended in HATmedium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum(Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 μg/mL gentamicin,2.5% Origen culturing supplement (Fisher), 10% 653-conditioned RPMI1640/Hepes media, 50 μM 2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μMaminopterin, and 16 μM thymidine) and then plated at 200 μL/well infifteen 96-well flat bottom tissue culture plates. The plates are thenplaced in a humidified 37° C. incubator containing 5% CO₂ and 95% airfor seven to ten days.

[0202] Detection of Human IgG Anti-Dengue virus Antibodies in MouseSerum

[0203] Solid phase EIA's can be used to screen mouse sera for human IgGantibodies specific for human Dengue virus. Briefly, plates can becoated with Dengue virus at 2 μg/mL in PBS overnight. After washing in0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blockedwith 1% (w/v) BSA in PBS, 200 μL/well for one hour at room temperature.Plates are used immediately or frozen at −20° C. for future use. Mouseserum dilutions are incubated on the Dengue virus coated plates at 50μL/well at room temperature for one hour. The plates are washed and thenprobed with 50 μL/well HRP-labeled goat 25 anti-human IgG, Fc specificdiluted 1:30,000 in 1% BSA-PBS for one hour at room temperature. Theplates can again be washed and 100 μL/well of the citrate-phosphatesubstrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01%H₂O₂ and 1 mg/mL OPD) is added for fifteen minutes at room temperature.Stop solution (4N sulfuric acid) is then added at 25 μL/well and theOD's are read at 490 nm via an automated plate spectrophotometer.

[0204] Detection of Completely Human Immunoglobulins in HybridomaSupernates

[0205] Growth positive hybridomas secreting fully human immunoglobulinscan be detected using a suitable EIA. Briefly, 96 well pop-out plates(VWR, 610744) can be coated with 10 μg/mL goat anti-human IgG Fc insodium carbonate buffer overnight at 4° C. The plates are washed andblocked with 1% BSA-PBS for one hour at 37° C. and used immediately orfrozen at −20° C. Undiluted hybridoma supernatants are incubated on theplates for one hour at 37° C. The plates are washed and probed with HRPlabeled goat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for onehour at 37° C. The plates are then incubated with substrate solution asdescribed above.

[0206] Determination of Fully Human Anti-Dengue Virus Reactivity

[0207] Hybridomas, as above, can be simultaneously assayed forreactivity to Dengue virus using a suitable RIA or other assay. Forexample, supernatants are incubated on goat anti-human IgG Fc plates asabove, washed and then probed with radio-labeled Dengue virus withappropriate counts per well for one hour at room temperature. The wellsare washed twice with PBS and bound radiolabeled Dengue virus isquantitated using a suitable counter.

[0208] Human anti-Dengue virus secreting hybridomas can be expanded incell culture and serially subcloned by limiting dilution. The resultingclonal populations can be expanded and cryopreserved in freezing medium(95% FBS, 5% DMSO) and stored in liquid nitrogen.

[0209] Isotyping

[0210] Isotype determination of the antibodies can be accomplished usingan EIA in a format similar to that used to screen the mouse immune serafor specific titers. Dengue virus can be coated on 96-well plates asdescribed above and purified antibody at 2 μg/mL can be incubated on theplate for one hour at room temperature. The plate is washed and probedwith HRP labeled goat anti-human IgG₁ or HRP labeled goat anti-humanIgG₃ diluted at 1:4000 in 1% BSA-PBS for one hour at room temperature.The plate is again washed and incubated with substrate solution asdescribed above.

[0211] Binding Kinetics of Human Anti-Dengue Virus Antibodies withDengue Virus

[0212] Binding characteristics for antibodies can be suitably assessedusing an Dengue virus capture EIA and BIAcore technology, for example.Graded concentrations of purified human Dengue virus antibodies can beassessed for binding to EIA plates coated with 2 μg/mL of Dengue virusin assays as described above. The OD's can be then presented as semi-logplots showing relative binding efficiencies.

[0213] Quantitative binding constants can be obtained, e.g., as follows,or by any other known suitable method. A BIAcore CM-5 (carboxymethyl)chip is placed in a BIAcore 2000 unit. HBS buffer (0.01 M HEPES, 0.15 MNaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over aflow cell of the chip at 5 μL/minute until a stable baseline isobtained. A solution (100 μL) of 15 mg of EDC(N-ethyl-N′-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200μL water is added to 100 μL of a solution of 2.3 mg ofNHS(N-hydroxysuccinimide) in 200 μL water. Forty (40) μL of theresulting solution is injected onto the chip. Six (6) μL of a solutionof human Dengue virus (15 μg/mL in 10 mM sodium acetate, pH 4.8) isinjected onto the chip, resulting in an increase of ca. 500 RU. Thebuffer is changed to TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 Msodium chloride, 2 mM calcium chloride, 2 mM magnesium acetate, 0.5%Triton X-100, 25 μg/mL BSA, pH 7.4) and flowed over the chip overnightto equilibrate it and to hydrolyze or cap any unreacted succinimideesters.

[0214] Antibodies are dissolved in the running buffer at 33.33, 16.67,8.33, and 4.17 nM. The flow rate is adjusted to 30 μL/min and theinstrument temperature to 25° C. Two flow cells are used for the kineticruns, one on which Dengue virus had been immobilized (sample) and asecond, underivatized flow cell (blank). 120 μL of each antibodyconcentration is injected over the flow cells at 30 μL/min (associationphase) followed by an uninterrupted 360 seconds of buffer flow(dissociation phase). The surface of the chip is regenerated (tissuenecrosis factor alpha/antibody complex dissociated) by two sequentialinjections of 30 μL each of 2 M guanidine thiocyanate. [0182] Analysisof the data is done using BIA evaluation 3.0 or CLAMP 2.0, as known inthe art. For each antibody concentration the blank sensogram issubtracted from the sample sensogram. A global fit is done for bothdissociation (k_(d), sec⁻¹) and association (k_(a), mol⁻¹ sec⁻¹) and thedissociation constant (K_(D), mol) calculated (k_(d)/k_(a)). Where theantibody affinity is high enough that the RUs of antibody capturedare >100, additional dilutions of the antibody are run.

[0215] Results and Discussion

[0216] Generation of Anti-Human Dengue Virus Monoclonal Antibodies

[0217] Several fusions are performed and each fusion is seeded infifteen plates (1440 wells/fusion) that yield several dozen antibodiesspecific for Dengue virus or Dengue virus-infected cells. Of these, someare found to consist of a combination of human and mouse Ig chains. Theremaining hybridomas secrete anti-Dengue virus antibodies consistingsolely of human heavy and light chains.

[0218] Binding Kinetics of Human Anti-Dengue Virus Antibodies

[0219] ELISA analysis confirms that purified antibody from most or allof these hybridomas bind Dengue virus in a concentration-dependentmanner. In this case, the avidity of the antibody for its cognateantigen (epitope) is measured. It should be noted that binding Denguevirus proteins directly to the EIA plate can cause denaturation of theprotein and the apparent binding affinities cannot be reflective ofbinding to undenatured protein. Fifty percent binding is found over arange of concentrations.

[0220] Quantitative binding constants are obtained using BIAcoreanalysis of the human antibodies and reveals that several of the humanmonoclonal antibodies are very high affinity with K_(D) in the range of1×10⁻⁹ to 7×10⁻¹². See FIG. 1, Panels A, B and C.

[0221] Conclusions

[0222] Several fusions are performed utilizing splenocytes from hybridmice containing human variable and constant region antibody transgenesthat are immunized with human Dengue virus. A set of several completelyhuman Dengue virus reactive IgG monoclonal antibodies of the IgG1isotype are generated. The completely human anti-Dengue virus antibodiesare further characterized. Several of generated antibodies have affinityconstants between 1×10⁹ and 9×10¹². The relatively high affinities ofthese fully human monoclonal antibodies make them suitable fortherapeutic applications in Dengue virus-dependent diseases, pathologiesor related conditions.

EXAMPLE 3 Isolation of Antibody by Phage Display of Human ImmuneRepertoire Reactive to Dengue Virus

[0223] The generation of antibodies from immune individuals as carriedout in this invention essentially involves four stages: (1) preparationof RNA from a source of antibody-producing cells, (2) reversetranscription and PCR amplification of heavy (Fd part) and light chains,(3) cloning of PCR inserts into a phagemid vector (pComb3) andexpression of a Fab library on the surface of phage, (4) panning of thelibrary against antigen to select specific Fabs. The antibodies selectedwill depend primarily on the RNA source, PCR amplification and theantigen used for panning.

[0224] Dengue Fab Antibody Isolation

[0225] Phage Library and Antibodies

[0226] Antibody phage-display libraries were prepared from RNA isolatedfrom human PBMC from a donor who recovered from infection with Denguevirus. The ability of serum obtained from the donor to neutralize Denguevirus in vitro is shown in Table 1. TABLE 1 Serum Dengue VirusNeutralization Titer Dengue Virus Serotype Sample 1 2 3 4 1 3980 1070 87170 2 300 170 220 20

[0227] The resulting library was panned for four consecutive rounds ofselection with increasing washing stringency. After the final round ofpanning, colonies were picked, expanded, and phage isolated. Specificbinders were identified in phage ELISA. Soluble Fab antibody wasprepared by digestion of isolated phagemid DNA which was digested toremove the cpIII gene, re-ligated, and used to transform E. coli.

[0228] Specifically, peripheral blood mononuclear cells (PBMC) wereisolated from blood of a human patient who had survived a Dengueinfection. The blood serum was lysed by vigorous mixing with denaturantsolution and RNA was prepared by adding 2 M sodium acetate (pH 4.0)lysate. The samples were extracted with acidic phenol (saturated with0.1 M citrate buffer [pH 4.3] [Sigma]) and a chloroformisoamyl alcoholmixture (24:1). After being incubated on ice for 15 minutes, the sampleswere centrifuged at 10,000×g for 20 minutes at 4° C. RNA wasprecipitated from the supernatant by the addition of 40 μg of glycogen(Boehringer Mannheim, Indianapolis, Ind.) and 15 ml of 2-propanol(Sigma), overnight incubation at −20° C., and centrifugation at 10,000×gfor 20 minutes at 4° C. The RNA pellet was re-dissolved in 3 ml ofdenaturant solution and re-precipitated for 3 hours at −20° C. after theaddition of an equal volume of 2-propanol. RNA was pelleted in amicrocentrifuge, washed twice with 70% ethanol, and re-suspended indiethylpyrocarbonate-treated water.

[0229] Library Construction

[0230] First-strand cDNA was prepared by priming with oligo d(T) with acDNA kit (Boehringer Mannheim) as recommended by the manufacturer. TheIgG1 Fd region and whole kappa and lambda light chains were thenamplified by PCR. Phage display libraries were constructed in the phagedisplay vector pComb3H. Briefly, the light-chain and heavy-chain PCRfragments were cloned into the SacI-XbaI and XhoI-SpeI restriction sitesof the phagemid, respectively. Ligation products were ethanolprecipitated and electroporated into Escherichia coli XLI-Blue cells(Stratagene, La Jolla, Calif.). The transformed E. coli cultures weregrown in SOC medium and then in SB medium containing 10 μg oftetracycline per ml and 20 μg of carbenicillin per ml, each for 1 hourat 37° C. The carbenicillin concentration was increased to 50 μg/ml, andafter the cells had grown for 1 hour, phage particle assembly wasinitiated by the addition of VCS-M13 helper phage (5×10¹¹ PFU). After anadditional 2 hours of culture, kanamycin was added to a concentration of50 μg/ml and the culture was grown overnight at 30° C. Phage wasrecovered from the cultures by removing bacteria by centrifugation at4,000×g and precipitating phage from the supernatant by addition of 4%polyethylene glycol and 0.5 M NaCl and incubation of the mixture on icefor 30 minutes. After centrifugation, phage pellets were re-suspended in500 μl of phosphate-buffered saline (PBS-4% nonfat dry milk (Bio-Rad,Hercules, Calif.) and centrifuged for 5 minutes in a microcentrifuge topellet bacterial debris.

[0231] Affinity Selection of Ab Libraries on Dengue Antigens

[0232] The Dengue antigens used for selection (panning) were NS1proteins obtained from a virus infected cell lysate.

[0233] A microtiter plate (Costar, Cambridge, Mass.) was coatedovernight at 4° C. with Dengue virus antigens. The plates were washedand blocked with 4% nonfat dry milk (Bio-Rad) for 1 hour at 37° C. Themilk solution was shaken out, phage solution was added to each well, andthe mixture was incubated for 2 hours at 37° C. on a rocker platform.The phage solution was removed, and the wells were washed. Bound phagewas eluted with glycine buffer (pH 2.2) and neutralized with 2 M Trisbase. Eluted phage was re-amplified for the next round of panning. Thelibraries were panned for four or five consecutive rounds withincreasing washing stringency (2, 5, and 10 wash steps thereafter, eachconsisting of a 5 minute incubation and vigorous pipetting). PhagemidDNA, isolated after the last round of panning, was digested with Nheland SpeI restriction endonucleases and re-ligated to excise the cpIIIgene and obtain plasmids producing soluble Fabs.

[0234] Screening of Soluble Fab Fragments

[0235] Microtiter wells were coated overnight at 4° C. with the twoDengue antigens used for panning and a control antigen, ovalbumin (4μg/ml) (Pierce, Rockford, Ill.). Soluble Fabs were tested by anenzyme-linked immunosorbent assay (ELISA). One of these Fabs wasdesignated Sid33, and was shown to bind to Dengue virus NS 1 protein.

[0236] DNA Sequencing

[0237] Fabs were analyzed for their DNA sequence with a 373A or 377Aautomated DNA sequencer (ABI, Foster City, Calif.), using a Taqfluorescent dideoxy terminator cycle-sequencing kit (ABI).

EXAMPLE 4 Generation of Human IgG Monoclonal Antibodies Reactive toDengue Virus

[0238] Expression and Purification of Antibody

[0239] The recombinant antibody DEN3, which is an IgG molecule, wasexpressed in the vector pDR12. The vector contains a light chain andheavy chain expression cassette in which transcription is driven from ahuman cytomegalovirus promoter. The heavy chain expression cassettecontains the genomic human IgG1 gene. Selection and amplification of theplasmid was done on the basis of expression of the gene for glutaminesynthetase. (See Bebbington et al., “High-level Expression of aRecombinant Antibody from Myeloma Cells Using a Glutamine SynthetaseGene as an Amplifiable Selectable Marker”, 10 BIO/TECHNOLOGY, 169-275(1992)).

[0240] DNA encoding the Fab Sid33 was cloned into the pDR12 expressionvector to produce pDRSid33, which places the Fab encoding sequences inthe framework of an IgG1 antibody molecule. The full IgG antibodymolecule that binds to Dengue virus NS 1 protein is referred to hereinas DEN3. pDRSid33 DNA was cut with Sal1 and transfected into Chinesehamster ovary cells (CHO-K1 cells; American Type Culture Collection,Manassas, Va.) using lipofectin reagent per the manufacturerrecommendations (Life Technologies, Grand Island, N.Y.). Cells weredistributed in six-well tissue culture treated plates and transfectedclones were selected with L-Methionine Sulfoximine ranging inconcentration from 40 to 140 μM (Sigma, St. Louis, Mo.).Post-transfection discrete colonies were assayed by enzyme-linkedimmunosorbent assay (ELISA) for antibody production. The highestproducers were cloned by limiting dilution, expanded and grown inthree-liter spinner flasks.

[0241] The recombinant DEN3 IgG1 monoclonal antibody was expressed inCHO-K1 cells in glutamine-free Glasgow minimum essential medium (GMEM,Sigma, St. Louis, Mo.) supplemented with 10% dialyzed fetal calf serum(FCS, Tissue Culture Biologicals, Tulare, Calif.), MEM non-essentialamino acids (Gibco-BRL, Grand Island, N.Y.), 1 mM MEM sodium pryuvate(Gibco-BRL), 500 μM L-glutamic acid, 500 μM L-asparagine, 30 μMadenosine, 30 μM adenosine, 30 μM guanosine, 30 μM cytidine, 30 μMuridine, 10 μM thymidine (Sigma), 100 U of penicillin/mL 100 μg ofstreptomycin/mL, and 50 methonine sulfoximine (Sigma) in a 3 literspinner flask. The supernatant was sterile filtered and purified overprotein A-Sepharose Fast Flow (Pharmacia, Arlington Heights, Ill.). Theantibody was eluted in 0.1 M citric acid, pH 3.0. The pH of the antibodysolution was immediately brought to neutrality by the addition of 1 MTris (pH 9.0), and the antibody was dialyzed against phosphate-bufferedsaline (PBS). Antibody concentrations were determined by absorbance at280 nm and confirmed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Antibody yields using this method rangedfrom 3 to 18 mg/Liter.

[0242] In anticipation of animal experiments care was taken to minimizecontamination with endotoxin, which was monitored using a quantitativechromagenic Limulus Amoebecyte Lysate assay (BioWhittaker, Walkersville,Md.) performed according to the manufacturer's recommendations. Whendetected, endotoxin was removed using polymyxin affinity columnchromatography (Bio-Rad, Hercules, Calif.).

EXAMPLE 5 Study Using Anti-Dengue Virus Antibodies and Controls toPrevent or Treat Dengue Virus Infection

[0243] Clinical Trials

[0244] Antibodies of the invention or fragments thereof may be tested inin vitro assays and animal models, and may be further evaluated forsafety, tolerance, and pharmacokinetics in groups of normal healthyadult volunteers. The volunteers are administered intramuscularly,intravenously or by an alternative delivery system a single dose of 0.5mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg or 15 mg/kg of an antibody or fragmentthereof which immunospecifically binds to a Dengue virus NS protein.Each volunteer is monitored at least 24 hours prior to receiving thesingle dose of the antibody or fragment thereof and each volunteer willbe monitored for at least 48 hours after receiving the dose at aclinical site. Then volunteers are monitored as outpatients on days 3,7, 14, 21, 28, 35, 42, 49, and 56 post-dose.

[0245] Blood samples are collected via an indwelling catheter or directvenipuncture using 10 ml Vacutainer tubes at the following intervals:(1) prior to administering the dose of the antibody or antibodyfragment; (2) during the administration of the dose of the antibody orantibody fragment; (3) 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 48hours after administering the dose of the antibody or antibody fragment;and (4) 3 days, 7 days 14 days, 21 days, 28 days, 35 days, 42 days, 49days, and 56 days after administering the dose of the antibody orantibody fragment. Samples are allowed to clot at room temperature andserum will be collected after centrifugation.

[0246] The antibody or antibody fragment is partially purified from theserum samples and the amount of antibody or antibody fragment in thesamples will be quantitated by ELISA. Briefly, the ELISA consists ofcoating microtiter plates overnight at 4° C. with an antibody thatrecognizes the antibody or antibody fragment administered to thevolunteer. The plates are then blocked for approximately 30 minutes atroom temperate with PBS-Tween-0.5% BSA. Standard curves are constructedusing purified antibody or antibody fragment, not administered to avolunteer. Samples are diluted in PBS-Tween-BSA. The samples andstandards are incubated for approximately 1 hour at room temperature.Next, the bound antibody is treated with a labeled antibody (e.g.,horseradish peroxidase conjugated goat-anti-human IgG) for approximately1 hour at room temperature. Binding of the labeled antibody is detected,e.g., by a spectrophotometer.

[0247] The concentration of antibody or antibody fragment levels in theserum of volunteers are corrected by subtracting the predose serum level(background level) from the serum levels at each collection intervalafter administration of the dose. For each volunteer the pharmacokineticparameters are computed according to the model-independent approach(Gibaldi et al. (Eds.), Pharmacokinetics, 2^(nd) Supplementary Edition,(Marcel Dekker, New York (1982)) from the corrected serum antibody orantibody fragment concentrations.

[0248] Treatment of Dengue Virus Infection in Humans Using anAnti-Dengue Virus NS Protein Antibody

[0249] The anti-Dengue Virus NS Protein antibody DEN3 may be used totreat patients infected with Dengue virus. Patients with the symptoms ofa Dengue virus infection will receive a single dose of either 0.1, 1.0,5.0 or 10 milligrams of DEN3 per kilogram bodyweight. Another group ofpatients will receive standard treatment for Dengue virus infection. TheDEN3 is administered as a single, intravenous infusion over a 60 minuteperiod. Clinical assessment, vital signs, and laboratory parameters aremeasured before, during and periodically for 28 days after the infusion.Accordingly, human treatment of Dengue virus infection in human patientsusing DEN3 is expected to provide a suitable treatment, including thereduction of severity of symptoms and duration of disease, as describedherein.

[0250] Equivalents

[0251] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

REFERENCES

[0252] 1. C. M. Rice, “Flaviviridae: The Viruses and Their Replication”,Fields Virology, 3^(rd) Edition, (Fields et al. (Eds.),Lippincott-Raven, Philadelphia, Pa. (1996)), 931-959.

[0253] 2. Wang et al., “Evolutionary Relationships of Endemic/Epidemicand Sylvatic Dengue Viruses”, 74 J. VIROL., 3227-3234 (2000).

[0254] 3. R. Rico-Hesse, “Molecular Evolution and Distribution of DengueViruses Type 1 and 2 in Nature”, 174 VIROLOGY, 479-493 (1990).

[0255] 4. B. L. Innis, “Antibody Responses to Dengue Virus Infection”,Dengue and Dengue Hemorrhagic Fever, (D. J. Gubler and G. Kuno (Eds.),CAB International, Cambridge (1997)) 221-243.

[0256] 5. Innis et al., “An Enzyme-Linked Immunosorbent Assay toCharacterize Dengue Infections Where Dengue and Japanese EncephalitisCo-Circulate”, 40 AM. J. TROP. MED. HYG., 418-427 (1989).

[0257] 6. A. B. Sabin, “Research on Dengue During World War II”, 1 AM.J. TROP. MED. HYG., 30-50 (1952).

[0258] 7. R. M. Myers and M. J. Varkey, “A Note on Sequential DengueInfection, Presumptive and Proved, with Report of an Instance of a ThirdProved Attack in One Individual”, 59 INDIAN J. MED. RES., 1231-1236(1971).

[0259] 8. Halstead et al., “Observations Related to Pathogenesis ofDengue Hemorrhagic Fever. IV: Relation of Disease Severity to AntibodyResponse and Virus Recovered”, 42 YALE J. BIOL. MED., 311-328 (1970).

[0260] 9. Kliks et al., “Evidence that Maternal Dengue Antibodies areImportant in the Development of Dengue Hemorrhagic in Infants”, 38 AM.J. TROP. MED. H YG., 411-419 (1988).

[0261] 10. Halstead et al., “Infant Dengue Hemorrahagic Fever: ResearchOpportunities Ignored”, EMERG. INFECT. DIS. (submitted 2002).

[0262] 11. S. B. Halstead, “Immunological Parameters of TogavirusDisease Syndromes”, The Togaviruses, Biology Structure, Replication, (R.W. Schlesinger (Ed.), Academic Press, New York, N.Y. (1980)) 107-173.

[0263] 12. S. B. Halstead, “Immune Enhancement of Viral Infection”, 31PROG. ALLERGY, 301-364 (1982).

[0264] 13. S. B. Halstead, “In Vivo Enhancement of Dengue VirusInfection in Rhesus Monkeys by Passively Transferred Antibody”, 140 J.INFECT. DIS., 527-533 (1979).

[0265] 14. Winter et al., “An Insular Outbreak of Dengue HemorrhagicFever. I: Epidemiologic Observations”, 17 AM. J. TROP. MED. HYG.,590-599 (1968).

[0266] 15. Burke et al., “A Prospective Study of Dengue Infections inBangkok”, 38 AM. J. TROP. MED. HYG., 172-180 (1988).

[0267] 16. Graham et al., “A Prospective Seroepidemiologic Study onDengue in Children Four to Nine Years of age in Yogyakarta, Indonesia.I: Studies in 1995-1996”, 61 AM. J. TROP. MED. HYG., 412-9 (1999).

[0268] 17. Sangkawibha et al., “Risk Factors in Dengue Shock Syndrome: AProspective Epidemiologic Study in Rayong, Thailand. I: The 1980Outbreak”, 120 AM. J. EPIDEMIOL., 653-669 (1984).

[0269] 18. Halstead et al., “Studies on the Pathogenesis of DengueInfection in Monkeys. II: Clinical Laboratory Responses to HeterologousInfection”, 128 J. INFECT. DIS., 15-22 (1973).

[0270] 19. Vaughn et al., “Dengue Viremia Titer, Antibody ResponsePattern, and Virus Serotype Correlate with Disease Severity”, 181 J.INFECT. DIS., 2-9 (2000).

[0271] 20. Halstead et al., “Heterogeneity of Infection Enhancement ofDengue 2 Strains by Monoclonal Antibodies”, 132 J. IMMUNOL., 1529-1532(1984).

[0272] 21. Halstead et al., “In Vitro Virulence Marker: Growth ofDengue-2 Virus in Human Leukocyte Suspension Cultures”, 31 INFECT.IMMUN., 102 (1981).

[0273] 22. D. M. Morens and S. B. Halstead, “Disease Severity-RelatedAntigenic Differences in Dengue 2 Strains Detected by Dengue 4Monoclonal Antibodies”, 22 J MED VIROL., 169-174 (1987).

[0274] 23. D. M. Morens and S. B Halstead, “Measurement ofAntibody-Dependent Infection Enhancement of Four Dengue Virus Serotypesby Monoclonal and Polyclonal Antibodies”, 71 J. GEN. VIROL., 2909-2914(1990).

[0275] 24. Kliks et al., “Antibody-Dependent Enhancement of Dengue VirusGrowth in Human Monocytes as a Risk Factor for Dengue HemorrhagicFever”, 40 AM. J. TROP. FILED HYG., 444-451 (1989).

[0276] 25. Watts et al., “Failure of Secondary Infection with AmericanGenotype Dengue 2 to Cause Dengue Haemorrhagic Fever”, [see comments],354 LANCET, 1431-4 (1999).

[0277] 26. Kochel et al., “Neutralization of American Genotype Dengue 2Viral Infection by Dengue 1 Antibodies May Have Prevented DengueHemorrhagic Fever in Iquitos, Peru”, LANCET [in press 2002].

[0278] 27. Guzman et al., “Do Escape Mutants Explain Rapid Increases inDengue Case-Fatality Rates within Epidemics?”, 355 LANCET, 1902-3(2000).

[0279] 28. S. B. Halstead and S. B. Russ, “Subclinical JapaneseEncephalitis. II: Antibody Responses of Americans to Single exposure toJE Virus”, 75 AM. J. HYG., 202-211 (1962).

[0280] 29. Guzman et al., “Enhanced Severity of Secondary Dengue 2Infections Occurring at an Interval of 20 Compared with 4 Years AfterDengue 1 Infection”, PANAMERICAN JOURNAL OF EPIDEMIOLOGY in press(2002).

1 4 1 444 DNA Artificial heavy chain 1 gccgccacca tggaatggag ctgggtctttctcttcttcc tgtcagtaac tacaggtgtc 60 cactcccagg ttcagctggt tcagtctggggctgaggtga agaagcctgg ggcctcagtg 120 aaggtttcct gcaaggcatc tggatacactttcaccaact actttctgca ctgggtgcga 180 caggcccccg gacaagggct tgagtggatgggaattatca agcctagtag tggtggtaca 240 accaacgcac agaagttcca gggcagagtcaccatgacca gggacacgtc cacgaacact 300 ttctacatgg agctgagcag cctgatatctgaggacacgg ccgtgtatta ctgtgcgcga 360 gaatccactc ccatatcagt ggccgacgactactacttcg gtatggacgt ctggggccaa 420 gggaccacgg tcaccgtgag ctca 444 2718 DNA Artificial light chain 2 aagcttacca tgggtgtgcc cactcaggtcctggggttgc tgctgctgtg gcttacagat 60 gccagatgtc agtccgtgct gactcagccaccctcagcgt ctgggacccc cgggcagagg 120 gtcaccatct cttgttctgg aagcacctccaacatcggaa gtaatactgt aaactggtac 180 cagcagctcc caggaacggc ccccaaactcctcatctata gtaatgatca gcggccctca 240 ggggtccctg accgattctc tggctccaagtctggcacct cagcctccct ggccatcagt 300 gggctccagt ctgaggatga ggctgattattactgtgcag catgggatga cagcctgaat 360 ggcctattcg gcggagggac caagctgaccgtcctaggtc agcccaaggc tgccccctcg 420 gtcactctgt tcccgccctc ctctgaggagcttcaagcca acaaggccac actggtgtgt 480 ctcataagtg acttctaccc gggagccgtgacagtggcct ggaaggcaca tagcagcccc 540 gtcaaggcgg gagtggagac caccacaccctccaaacaaa gcaacaacaa gtacgcggcc 600 agcagctacc tgagcctgac gcctgagcagtggaagtccc acagaagcta cagctgccag 660 gtcacgcatg aagggagcac cgtggagaagacagtgcgcc cctacagaat gttcataa 718 3 148 PRT Artificial heavy chain 3Ala Ala Thr Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val 1 5 1015 Thr Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 2530 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 4045 Tyr Thr Phe Thr Asn Tyr Phe Leu His Trp Val Arg Gln Ala Pro Gly 50 5560 Gln Gly Leu Glu Trp Met Gly Ile Ile Lys Pro Ser Ser Gly Gly Thr 65 7075 80 Thr Asn Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 8590 95 Ser Thr Asn Thr Phe Tyr Met Glu Leu Ser Ser Leu Ile Ser Glu Asp100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ser Thr Pro Ile Ser ValAla 115 120 125 Asp Asp Tyr Tyr Phe Gly Met Asp Val Trp Gly Gln Gly ThrThr Val 130 135 140 Thr Val Ser Ser 145 4 238 PRT Artificial light chain4 Lys Leu Thr Met Gly Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu 1 5 1015 Trp Leu Thr Asp Ala Arg Cys Gln Ser Val Leu Thr Gln Pro Pro Ser 20 2530 Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser 35 4045 Thr Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro 50 5560 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asp Gln Arg Pro Ser 65 7075 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 8590 95 Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys100 105 110 Ala Ala Trp Asp Asp Ser Leu Asn Gly Leu Phe Gly Gly Gly ThrLys 115 120 125 Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val ThrLeu Phe 130 135 140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala ThrLeu Val Cys 145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val ThrVal Ala Trp Lys Ala 165 170 175 Asp Ser Ser Pro Val Lys Ala Gly Val GluThr Thr Thr Pro Ser Lys 180 185 190 Gln Ser Asn Asn Lys Tyr Ala Ala SerSer Tyr Leu Ser Leu Thr Pro 195 200 205 Glu Gln Trp Lys Ser His Arg SerTyr Ser Cys Gln Val Thr His Glu 210 215 220 Gly Ser Thr Val Glu Lys ThrVal Ala Pro Thr Glu Cys Ser 225 230 235

What is claimed is:
 1. At least one isolated mammalian anti-Dengue virusantibody, comprising at least one variable region comprising the aminoacid sequence set forth in SEQ ID NOS: 3 or
 4. 2. An antibody accordingto claim 1, wherein said antibody binds Dengue virus NS-1 protein.
 3. Ananti-Dengue virus antibody according to claim 1, wherein said antibodybinds at least one Dengue virus NS protein.
 4. An isolated nucleic acidencoding at least one mammalian anti-Dengue virus antibody having atleast one variable region comprising the amino acid sequence set forthin SEQ ID NOS: 3 or
 4. 5. An isolated nucleic acid vector comprising anisolated nucleic acid according to claim
 4. 6. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 5. 7. A host cell according to claim 6, wherein said host cell isat least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or anyderivative, immortalized or transformed cell thereof.
 8. A method forproducing at least one anti-Dengue virus antibody, comprisingtranslating a nucleic acid according to claim 4 under conditions invitro, in vivo or in situ, such that the anti-Dengue virus antibody isexpressed in detectable or recoverable amounts.
 9. A compositioncomprising at least one isolated mammalian anti-Dengue virus antibodyhaving at least one variable region comprising an amino acid sequenceset forth in SEQ ID NOS: 3 or 4, and at least one pharmaceuticallyacceptable carrier or diluent.
 10. A composition according to claim 9,further comprising at least one composition comprising an effectiveamount of at least one compound or protein selected from at least one ofa detectable label or reporter, a Dengue virus replication antagonist, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, an antimicrobial, a corticosteriod, anerythropoietin, an antigen for immunization, an immunoglobulin, a growthhormone, a hormone replacement drug, a radiopharmaceutical, an asthmamedication, an inhaled steroid, an epinephrine or analog, a cytokine, ora cytokine antagonist.
 11. A method for diagnosing or treating a Denguevirus-related condition in a cell, tissue, organ, patient or animal,comprising: (a) contacting or administering a composition comprising aneffective amount of at least one isolated mammalian anti-Dengue virusantibody having at least one variable region comprising SEQ ID NOS: 3 or4, with, or to, said cell, tissue, organ, patient or animal.
 12. Amethod according to claim 11, wherein said effective amount is 0.001 to50 mg/kilogram of said cells, tissue, organ, patient or animal.
 13. Amethod according to claim 11, wherein said contacting or saidadministrating is by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.14. A method according to claim 11, further comprising administering,prior, concurrently or after said (a) contacting or administering, atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a Dengue virus replication antagonist, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, an antimicrobial, a corticosteriod, anerythropoietin, an antigen for immunization, an immunoglobulin, a growthhormone, a hormone replacement drug, a radiopharmaceutical, an asthmamedication, an inhaled steroid, an epinephrine or analog, a cytokine, ora cytokine antagonist.
 15. A medical device, comprising at least oneisolated mammalian anti-Dengue virus antibody having at least onevariable region comprising SEQ ID NOS: 3 or 4, wherein said device issuitable to contacting or administering said at least one anti-Denguevirus antibody by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.16. An article of manufacture for human pharmaceutical or diagnosticuse, comprising packaging material and a container comprising a solutionor a lyophilized form of at least one isolated mammalian anti-Denguevirus antibody having at least one variable region comprising SEQ IDNOS: 3 or
 4. 17. The article of manufacture of claim 16, wherein saidcontainer is a component of a parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.
 18. A method for producing at least one isolatedmammalian anti-Dengue virus antibody having at least one variable regioncomprising SEQ ID NOS: 3 or 4, comprising providing a host cell ortransgenic animal or transgenic plant or plant cell capable ofexpressing in recoverable amounts said antibody.
 19. At least oneanti-Dengue virus antibody produced by a method according to claim 18.20. At least one isolated mammalian anti-Dengue virus antibody,comprising either (i) all of the heavy chain complementarity determiningregions (CDR) amino acid sequences of SEQ ID NO: 3; or (ii) all of thelight chain CDR amino acids sequences of SEQ ID NO:
 4. 21. A Denguevirus antibody according to claim 20, wherein said antibody binds aDengue virus NS protein.
 22. A Dengue virus antibody according to claim20, wherein said antibody binds at least one Dengue virus NS 1 protein.23. An isolated nucleic acid encoding at least one isolated mammaliananti-Dengue virus antibody either (i) all of the heavy chain CDR aminoacid sequences of SEQ ID NO: 3; or (ii) all of the light chain CDR aminoacids sequences of SEQ ID NO:
 4. 24. An isolated nucleic acid vectorcomprising an isolated nucleic acid according to claim 4 having thenucleotide sequence set forth in SEQ ID NO: 1 or
 2. 25. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 24. 26. A host cell according to claim 25, wherein said host cellis at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1,Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or anyderivative, immortalized or transformed cell thereof.
 27. A method forproducing at least one anti-Dengue virus antibody, comprisingtranslating a nucleic acid according to claim 23 under conditions invitro, in vivo or in situ, such that the Dengue virus antibody isexpressed in detectable or recoverable amounts.
 28. A compositioncomprising at least one isolated mammalian anti-Dengue virus antibodyhaving either (i) all of the heavy chain CDR amino acid sequences of SEQID NO: 3; or (ii) all of the light chain CDR amino acids sequences ofSEQ ID NO: 4, and at least one pharmaceutically acceptable carrier ordiluent.
 29. A composition according to claim 28, further comprising atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a Dengue virus replication antagonist, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, an antimicrobial, a corticosteriod, anerythropoietin, an antigen for immunization, an immunoglobulin, a growthhormone, a hormone replacement drug, a radiopharmaceutical, an asthmamedication, an inhaled steroid, an epinephrine or analog, a cytokine, ora cytokine antagonist.
 30. A method for diagnosing or treating a Denguevirus-related condition in a cell, tissue, organ, patient, animal orpopulation of subjects comprising: (a) contacting or administering acomposition comprising an effective amount of at least one isolatedmammalian anti-Dengue virus antibody having either (i) all of the heavychain CDR amino acid sequences of SEQ ID NO: 3; or (ii) all of the lightchain CDR amino acids sequences of SEQ ID NO: 4, with, or to, said cell,tissue, organ, patient or animal.
 31. A method according to claim 30,wherein said effective amount is 0.001 to 50 mg/kilogram of said cells,tissue, organ, patient or animal.
 32. A method according to claim 30,wherein said contacting or said administrating is by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 33. Amethod according to claim 30, further comprising administering, prior,concurrently or after said (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aDengue virus replication antagonist, a non-steroid anti-inflammatorydrug (NSAID), an analgesic, an anesthetic, a sedative, a localanesthetic, an antimicrobial, a corticosteriod, an erythropoietin, anantigen for immunization, an immunoglobulin, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an asthma medication, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 34. A medical device, comprising at least one isolatedmammalian anti-Dengue virus antibody having either (i) all of the heavychain CDR amino acid sequences of SEQ ID NO: 3; or (ii) all of the lightchain CDR amino acids sequences of SEQ ID NO: 4, wherein said device issuitable to contacting or administering said at least one anti-Denguevirus antibody by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.35. An article of manufacture for human pharmaceutical or diagnosticuse, comprising packaging material and a container comprising a solutionor a lyophilized form of at least one isolated mammalian anti-Denguevirus antibody having either (i) all of the heavy chain CDR amino acidsequences of SEQ ID NO: 3; or (ii) all of the light chain CDR aminoacids sequences of SEQ ID NO:
 4. 36. The article of manufacture of claim35, wherein said container is a component of a parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermaldelivery device or system.
 37. A method for producing at least oneisolated mammalian anti-Dengue virus antibody having either (i) all ofthe heavy chain CDR amino acid sequences of SEQ ID NO: 3; or (ii) all ofthe light chain CDR amino acids sequences of SEQ ID NO: 4, comprisingproviding a host cell or transgenic animal or transgenic plant or plantcell capable of expressing in recoverable amounts nucleic acid moleculesencoding said antibody.
 38. At least one anti-Dengue virus antibodyproduced by a method according to claim
 37. 39. At least one isolatedmammalian anti-Dengue virus antibody, comprising at least one heavychain or light chain CDR having the amino acid sequence of at least oneof SEQ ID NOS: 3 or
 4. 40. A Dengue virus antibody according to claim39, wherein said antibody binds a Dengue virus NS protein.
 41. A Denguevirus antibody according to claim 39, wherein said antibody binds atleast one Dengue virus NS 1 protein.
 42. An isolated nucleic acidencoding at least one isolated mammalian anti-Dengue virus antibodyhaving at least one heavy chain or light chain CDR having the amino acidsequence of at least one of SEQ ID NOS: 3 or
 4. 43. An isolated nucleicacid vector comprising an isolated nucleic acid according to claim 42.44. A prokaryotic or eukaryotic host cell comprising an isolated nucleicacid according to claim
 43. 45. A host cell according to claim 44,wherein said host cell is at least one selected from COS-1, COS-7,HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, orlymphoma cells, or any derivative, immortalized or transformed cellthereof.
 46. A method for producing at least one anti-Dengue virusantibody, comprising translating a nucleic acid according to claim 42under conditions in vitro, in vivo or in situ, such that the Denguevirus antibody is expressed in detectable or recoverable amounts.
 47. Acomposition comprising at least one isolated mammalian anti-Dengue virusantibody having at least one heavy chain or light chain CDR having theamino acid sequence of at least one of SEQ ID NOS: 3 or 4, and at leastone pharmaceutically acceptable carrier or diluent.
 48. A compositionaccording to claim 47, further comprising at least one compositioncomprising an effective amount of at least one compound or proteinselected from at least one of a detectable label or reporter, a Denguevirus replication antagonist, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, anantimicrobial, a corticosteriod, an erythropoietin, an antigen forimmunization, an immunoglobulin, a growth hormone, a hormone replacementdrug, a radiopharmaceutical, an asthma medication, an inhaled steroid,an epinephrine or analog, a cytokine, or a cytokine antagonist.
 49. Amethod for diagnosing or treating a Dengue virus related condition in acell, tissue, organ, patient or animal, comprising: (a) contacting oradministering a composition comprising an effective amount of at leastone isolated mammalian anti-Dengue virus antibody having at least oneheavy chain or light chain CDR having the amino acid sequence of atleast one of SEQ ID NOS: 3 or 4, with, or to, said cell, tissue, organ,patient or animal.
 50. A method according to claim 49, wherein saideffective amount is 0.001 to 50 mg/kilogram of said cells, tissue,organ, patient or animal.
 51. A method according to claim 49, whereinsaid contacting or said administrating is by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 52. Amethod according to claim 49, further comprising administering, prior,concurrently or after said (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aDengue virus replication antagonist, a non-steroid anti-inflammatorydrug (NSAID), an analgesic, an anesthetic, a sedative, a localanesthetic, an antimicrobial, a corticosteriod, an erythropoietin, anantigen for immunization, an immunoglobulin, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an asthma medication, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 53. A medical device, comprising at least one isolatedmammalian anti-Dengue virus antibody having at least one heavy chain orlight chain CDR having the amino acid sequence of at least one of SEQ IDNOS: 3 or 4, wherein said device is suitable to contacting oradministering said at least one anti-Dengue virus antibody by at leastone mode selected from parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 54. Anarticle of manufacture for human pharmaceutical or diagnostic use,comprising packaging material and a container comprising at least oneisolated mammalian anti-Dengue virus antibody or nucleic acid moleculeencoding said antibody, having at least one heavy chain or light chainCDR having the amino acid sequence of at least one of SEQ ID NOS: 3 or4.
 55. The article of manufacture of claim 54, wherein said container isa component of a parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.
 56. A method for producing at least one isolatedmammalian anti-Dengue virus antibody having at least one heavy chain orlight chain CDR having the amino acid sequence of at least one of SEQ IDNOS: 3 or 4, comprising providing a host cell or transgenic animal ortransgenic plant or plant cell capable of expressing a nucleic acidmolecule encoding said antibody in recoverable amounts.
 57. At least oneanti-Dengue virus antibody produced by a method according to claim 56wherein said nucleic acid molecule comprises SEQ ID NO: 1 or
 2. 58. Atleast one isolated mammalian anti-Dengue virus antibody that binds tothe same region of a Dengue virus protein as an antibody comprising atleast one heavy chain or light chain CDR having the amino acid sequenceof at least one of SEQ ID NOS: 3 or
 4. 59. A Dengue virus antibodyaccording to claim 58, wherein said antibody binds a Dengue virus NSprotein.
 60. A Dengue virus antibody according to claim 58, wherein saidantibody substantially neutralizes at least one activity of at least oneDengue virus protein.
 61. An isolated nucleic acid encoding at least oneisolated mammalian anti-Dengue virus antibody that binds to the sameregion of a Dengue virus protein as an antibody comprising at least oneheavy chain or light chain CDR having the amino acid sequence of atleast one of SEQ ID NOS: 3 or
 4. 62. An isolated nucleic acid vectorcomprising an isolated nucleic acid according to claim
 61. 63. Aprokaryotic or eukaryotic host cell comprising an isolated nucleic acidaccording to claim
 62. 64. A host cell according to claim 63, whereinsaid host cell is at least one selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. 65.A method for producing at least one anti-Dengue virus antibody,comprising translating a nucleic acid according to claim 61 underconditions in vitro, in vivo or in situ, such that the Dengue virusantibody is expressed in detectable or recoverable amounts.
 66. Acomposition comprising at least one isolated mammalian anti-Dengue virusantibody that binds to the same region of a Dengue virus protein as anantibody comprising at least one heavy chain or light chain CDR havingthe amino acid sequence of at least one of SEQ ID NOS: 3 or 4, and atleast one pharmaceutically acceptable carrier or diluent.
 67. Acomposition according to claim 66, further comprising at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aDengue virus replication antagonist, a non-steroid anti-inflammatorydrug (NSAID), an analgesic, an anesthetic, a sedative, a localanesthetic, an antimicrobial, a corticosteriod, an erythropoietin, anantigen for immunization, an immunoglobulin, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an asthma medication, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 68. A method for diagnosing or treating a Dengue virusrelated condition in a cell, tissue, organ, patient or animal,comprising: (a) contacting or administering a composition comprising aneffective amount of at least one isolated mammalian anti-Dengue virusantibody that binds to the same region of a Dengue virus protein as anantibody comprising at least one heavy chain or light chain CDR havingthe amino acid sequence of at least one of SEQ ID NOS: 3 or 4, with, orto, said cell, tissue, organ, patient or animal.
 69. A method accordingto claim 68, wherein said effective amount is 0.001 to 50 mg/kilogram ofsaid cells, tissue, organ, patient or animal.
 70. A method according toclaim 68, wherein said contacting or said administrating is by at leastone mode selected from parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 71. Amethod according to claim 68, further comprising administering, prior,concurrently or after said step (a) contacting or administering, atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a Dengue virus replication antagonist, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, an antimicrobial, a corticosteriod, anerythropoietin, an antigen for immunization, an immunoglobulin, a growthhormone, a hormone replacement drug, a radiopharmaceutical, an asthmamedication, an inhaled steroid, an epinephrine or analog, a cytokine, ora cytokine antagonist.
 72. A medical device, comprising at least oneisolated mammalian anti-Dengue virus antibody or nucleic acid moleculeencoding said antibody that binds to the same region of a Dengue virusprotein as an antibody comprising at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:3 or 4, wherein said device is suitable to contacting or administeringsaid at least one anti-Dengue virus antibody by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 73. Anarticle of manufacture for human pharmaceutical or diagnostic use,comprising packaging material and a container comprising at least oneisolated mammalian anti-Dengue virus antibody or nucleic acid moleculeencoding said antibody that binds to the same region of a Dengue virusprotein as an antibody comprising at least one heavy chain or lightchain CDR having the amino acid sequence of at least one of SEQ ID NOS:3 or
 4. 74. The article of manufacture of claim 73, wherein saidcontainer is a component of a parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.
 75. A method for producing at least one isolatedmammalian anti-Dengue virus antibody that binds to the same region of aDengue virus protein as an antibody comprising at least one heavy chainor light chain CDR having the amino acid sequence of at least one of SEQID NOS: 3 or 4, comprising providing a host cell or transgenic animal ortransgenic plant or plant cell capable of expressing in recoverableamounts said antibody.
 76. At least one anti-Dengue virus antibodyproduced by a method according to claim
 75. 77. At least one isolatedmammalian anti-Dengue virus antibody, comprising at least one human CDR,wherein said antibody specifically binds at least one epitope comprisingat least 1-3, to the entire amino acid sequence of a Dengue virus NSprotein.
 78. A Dengue virus antibody according to claim 77, wherein saidantibody binds a Dengue virus NS 1 protein.
 79. A Dengue virus antibodyaccording to claim 77, wherein said antibody substantially neutralizesat least one activity of at least one Dengue virus NS protein.
 80. Anisolated nucleic acid encoding at least one isolated mammaliananti-Dengue virus antibody having at least one human CDR, wherein saidantibody specifically binds at least one epitope comprising two or moreamino acids of Dengue virus NS 1 protein.
 81. An isolated nucleic acidvector comprising an isolated nucleic acid according to claim
 80. 82. Aprokaryotic or eukaryotic host cell comprising an isolated nucleic acidaccording to claim
 81. 83. A host cell according to claim 82, whereinsaid host cell is at least one selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. 84.A method for producing at least one anti-Dengue virus antibody,comprising translating a nucleic acid according to claim 80 underconditions in vitro, in vivo or in situ, such that the Dengue virusantibody is expressed in detectable or recoverable amounts, wherein saidnucleic acid molecule comprises SEQ ID NO: 1 or
 2. 85. A compositioncomprising at least one isolated mammalian anti-Dengue virus antibodyhaving at least one human CDR, wherein said antibody specifically bindsat least one epitope comprising two or more amino acids of Dengue virusNS 1 protein, and at least one pharmaceutically acceptable carrier ordiluent.
 86. A composition according to claim 85, further comprising atleast one composition comprising an effective amount of at least onecompound or protein selected from at least one of a detectable label orreporter, a Dengue virus replication antagonist, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, an antimicrobial, a corticosteriod, anerythropoietin, an antigen for immunization, an immunoglobulin, a growthhormone, a hormone replacement drug, a radiopharmaceutical, an asthmamedication, an inhaled steroid, an epinephrine or analog, a cytokine, ora cytokine antagonist.
 87. A method for diagnosing or treating a Denguevirus related condition in a cell, tissue, organ, patient or animal,comprising: (a) contacting or administering a composition comprising aneffective amount of at least one isolated mammalian anti-Dengue virusantibody having at least one human CDR, wherein said antibodyspecifically binds at least one epitope comprising two or more aminoacids of a Dengue virus NS 1 protein, with, or to, said cell, tissue,organ, patient or animal.
 88. A method according to claim 87, whereinsaid effective amount is 0.001 to 50 mg/kilogram of said cells, tissue,organ, patient or animal.
 89. A method according to claim 87, whereinsaid contacting or said administrating is by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
 90. Amethod according to claim 87, further comprising administering, prior,concurrently or after said (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aDengue virus replication antagonist, a non-steroid anti-inflammatorydrug (NSAID), an analgesic, an anesthetic, a sedative, a localanesthetic, an antimicrobial, a corticosteriod, an erythropoietin, anantigen for immunization, an immunoglobulin, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an asthma medication, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 91. A medical device, comprising at least one isolatedmammalian anti-Dengue virus antibody or a nucleic acid molecule encodingsaid antibody, having at least one human CDR, wherein said antibodyspecifically binds at least one epitope comprising at least 1-3, to theentire amino acid sequence of SEQ ID NOS: 3 and 4, wherein said deviceis suitable to contacting or administering said at least one anti-Denguevirus antibody by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.92. An article of manufacture for human pharmaceutical or diagnosticuse, comprising packaging material and a container comprising at leastone isolated mammalian anti-Dengue virus antibody or a nucleic acidmolecule encoding said antibody, having at least one human CDR, whereinsaid antibody specifically binds at least one epitope comprising two ormore amino acids of Dengue virus NS1 protein.
 93. The article ofmanufacture of claim 92, wherein said container is a component of aparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery device or system.
 94. A method for producing atleast one isolated mammalian anti-Dengue virus antibody having at leastone human CDR, wherein said antibody specifically binds at least oneepitope comprising two or more amino acids of Dengue virus NS1 protein,comprising providing a host cell or transgenic animal or transgenicplant or plant cell capable of expressing in recoverable amounts saidantibody.
 95. At least one anti-Dengue virus antibody produced by amethod according to claim 94.